Protease-resistant systems for polypeptide display and methods of making and using thereof

ABSTRACT

The present invention generally relates to bacterial polypeptide display systems, libraries using these bacterial display systems, and methods of making and using these systems, including methods for improved display of polypeptides on the extracellular surface of bacteria using circularly permuted transmembrane bacterial polypeptides that have been modified to increase resistance to protease degradation and to enhance polypeptide display characteristics.

This application claims the benefit of U.S. Provisional Application No.61/682,164, filed Aug. 10, 2012, which is incorporated herein byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “42652_520001USSeqList.txt”, whichwas created on May 2, 2014 and is 134 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to bacterial polypeptide displaysystems, to libraries using these bacterial display systems, and tomethods of making and using these systems, including methods forimproved display of polypeptides on the extracellular surface ofbacteria using circularly permuted transmembrane bacterial polypeptidesthat have been modified to increase resistance to protease degradationand to enhance polypeptide display characteristics.

BACKGROUND OF THE INVENTION

Polypeptide display technologies have substantially impacted basic andapplied research applications ranging from drug discovery to materialssynthesis. Previous expression vectors for polypeptide display librariesusing host cells suffer from a variety of problems. The problems of theprior art methods include (1) only small peptides may be expressed, (2)large libraries cannot be selected, (3) the polypeptides are notexpressed on the outer membrane surface, but are instead expressed inthe periplasmic space between the inner and the outer membranes, (4)polypeptides that are displayed on the outer membrane surface do notproperly bind or interact with large molecules and certain targets, and(5) analyzing expression on fimbriae or flagella results in loss of somedesired polypeptides due to mechanical shearing.

Protein display on the surface of bacterial cells holds the potential tosimplify and accelerate the process of ligand isolation sinceexperimental procedures with bacteria are efficient, and screening canbe performed using FACS. Although several different bacterial displaysystems have been reported, their usefulness has been restricted bytechnical limitations including accessibility on the cell surface,inability to display highly diverse sequences, adverse effects on cellgrowth and viability, and difficulty in expressing long polypeptides. Inaddition, utility has been hampered by protease sensitivity of systemswhen exposed to complex mixtures that include proteases.

Thus, a need exists for a more robust display methodology that requiresminimal technical expertise, is less labor intensive, and speeds theprocess of ligand isolation from weeks to days as compared to priormethods.

SUMMARY OF THE INVENTION

The present invention relates to carrier polypeptides (CPs) and usesthereof. As used herein, the term carrier polypeptide refers to atransmembrane polypeptide that is designed to display a molecule,referred to herein as display moiety or displayed moiety (DM), on eitheror both of the N- and C-termini of the CP. In some embodiments, the CPis barrel-shaped beta sheet transmembrane polypeptide having proteaseresistant sequences in at least one extracellular region of thepolypeptide, such as in an extracellular loop of the polypeptide or atthe N- and/or C-termini of the CP. In some embodiments, the CP is acircularly permuted beta barrel-shaped beta transmembrane polypeptidehaving glycine-serine rich sequences, or other flexible peptidesequences, at the N- and/or C-termini of the CP or in otherextracellular regions of the CP. CPs provided herein include carrierpolypeptides referred to herein as “CYTX-CPs.”

The CYTX-CP includes at least the amino acid sequence: YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSF TYT (SEQ IDNO: 1) or an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 1.

In some embodiments, the CYTX-CP includes at least the amino acidsequence: YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAG (SEQ ID NO: 56) or an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 56.

In some embodiments, the CYTX-CP includes at least the amino acidsequence: YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAG (SEQ ID NO: 57) or an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 57.

The invention also provides polypeptide display platforms (DPs) thatinclude (i) one or more carrier polypeptides (CPs) having at least atransmembrane portion and N- and C-termini that are exposed when the CPis displayed on the outer membrane of a replicable biological entity,and (ii) one or more displayed moieties (DMs), such as for example, atleast one polypeptide or other biological molecule. Suitable DMsinclude, by way of non-limiting example, a substrate sequence (S), e.g.,a peptide sequence that is cleaved by one or more proteases; a maskingmoiety (MM), e.g., a peptide sequence that reduces the ability of anantibody or antibody fragment (AB) to bind a target; an exosite (EX); anallosteric binding site (AS); an antibody or antibody fragment (AB); areceptor (R); a ligand (L); an inhibitor (I); and any combinationthereof. In some embodiments, CPs having only one DM have the generalstructural arrangement from N-terminus to C-terminus as follows: DM-CPor CP-DM.

In some embodiments, CPs having only one DM include, for example, DPshaving a structural arrangement from N-terminus to C-terminus such asMM-CP, CP-MM; S-CP, CP-S; EX-CP, CP-EX; AB-CP, CP-AB, AS-CP, CP-AS,R-CP, CP-R, L-CP, CP-L, I-CP, or CP-I. In some embodiments, the CPincludes at least the amino acid sequence of SEQ ID NO: 1. In someembodiments, the CP includes at least the amino acid sequence of SEQ IDNO: 56. In some embodiments, the CP includes at least the amino acidsequence of SEQ ID NO: 57.

In some embodiments, the DM is a masking moiety (MM), e.g., a peptidesequence that reduces the ability of an antibody or antibody fragment(AB) to bind a target. In some embodiments, the target is a targetselected from those shown in Table 1.

In some embodiments, the DM is a substrate sequence (S), e.g., a peptidesequence that is cleaved by one or more proteases. In some embodiments,the protease is a protease selected from those shown in Table 2.

The DP can also include additional elements, including, by way ofnon-limiting example, a tag (T), such as a protease resistant N-terminaltag, a protease-resistant C-terminal tag or both N-terminal andC-terminal protease resistant tags (e.g., T1 and T2). In someembodiments, the DP includes an N-terminal tag that includes the aminoacid sequence EYMPME (SEQ ID NO: 8). In some embodiments, the DPincludes a C-terminal tag that includes a histidine tag, such as, forexample, an 8-His tag (HHHHHHHH, SEQ ID NO: 13). In some embodiments,the DP includes an N-terminal tag that includes the amino acid sequenceEYMPME (SEQ ID NO: 8) and a C-terminal tag that includes an 8-HIS tag(HHHHHHHH, SEQ ID NO: 13).

These DP embodiments have the general structural arrangement fromN-terminus to C-terminus as follows: T1-DM-CP-T2 or T1-CP-DM-T2, whereDM can be any suitable displayed molecule, such as, for example, asubstrate, an antibody or fragment thereof, or a masking moiety.Additional non-limiting examples of DPs include T1-DM-CP, DM-CP-T2,T1-CP-DM, and CP-DM-T2.

In some embodiments, the DP includes more than one DM. In someembodiments, DM is a combination of displayed moieties, such as, forexample, MM-AB or AB-MM; MM-S-AB or AB-S-MM, S-AB or AB-S such that theDP has the structural arrangement such as, e.g., T1-DM1-DM2-CP-T2 orT1-CP-DM1-DM2-T2; T1-DM1-DM2-DM3-CP-T2 or T1-CP-DM1-DM2-DM3-T2 and soon. In some embodiments, the DP includes at least two DM and each DMneed not be adjacent to each other. For example, the DP can include thestructural arrangement DM1-T1-DM2-CP-T2 or T1-CP-DM1-T2-DM2; or the DPcan include the structural arrangement T1-DM1-CP-DM2-T2, such as, forexample, EX-T1-S-CP-T2 or T1-CP-S-T2-EX; or T1-S-CP-EX-T2 orT1-EX-CP-S-T2.

In some embodiments, the DP can also include one or more linkers betweentwo adjacent elements within the DP. For example, in some embodiments,the DP includes one or more of the following: a linker between theN-terminal tag and the DM, a linker between the DM and transmembraneportion of the CP, a linker between the transmembrane portion of the CPand the C-terminal tag. Examples of DPs with linkers in a variety oflocations are shown in FIGS. 23-25 and 29-31.

The invention also provides replicable biological entities (RBEs) thatexpress one or more polypeptide DPs on the outer surface, e.g., outermembrane or extracellular surface, of the RBE. These RBE are used tocreate libraries of candidate DMs for evaluation, screening and otheranalytical assessment. In some embodiments, the replicable biologicalentity is a bacterial cell, a yeast cell or a mammalian cell. In someembodiments, the replicable biological entity is a bacterial cell. Insome embodiments, the bacterial cell is Escherichia coli, Shigellasonnei, Shigella dysenteriae, Shigella flexneri, Salmonella typhii,Salmonella typhimurium, Salmonella enterica, Enterobacter aerogenes,Serratia marcescens, Yersinia pestis, Bacillus cereus, Bacillussubtilis, or Klebsiella pneumoniae. RBEs of the embodiments typicallydisplay mature DPs in their outer surface. RBEs of the embodiments alsoinclude DPs with signal sequences as well as nucleic acid sequencesencoding such mature DPs and signal sequence-containing DPsintracellularly.

The invention also provides nucleic acids and/or expression vectorsencoding one or more CPs and/or one or more DPs, where the CP and/or DPis/are designed to be more protease-resistant, to allow for the displayof larger polypeptides, and/or to exhibit improved displaycharacteristics.

Previous carrier proteins using different circularly permutedpolypeptides are described, for example, in PCT Publication No. WO2005/047461; PCT Publication No. WO 2009/014726; U.S. Pat. No.8,361,933; U.S. Pat. No. 8,293,685; U.S. Pat. No. 7,256,038; U.S. Pat.No. 7,612,019; U.S. Patent Application Publication No. 2010/0113303; PCTPublication No. WO 2007/027935; U.S. Pat. No. 7,666,817; U.S. PatentApplication Publication No. 20100173349, and US Patent ApplicationPublication No. 20130123141, each of which is hereby incorporated byreference in its entirety.

Previous methods of identifying and using protease cleavage sites withina displayed polypeptide to allow for selective cleavage of a detectablemoiety, e.g., peptide substrate, in a specific disease microenvironmentare described, for example, in PCT Publication No. WO 2005/047461; PCTPublication No. WO 2009/014726; U.S. Pat. No. 8,361,933; U.S. Pat. No.8,293,685; U.S. Pat. No. 7,256,038; U.S. Pat. No. 7,612,019; U.S. PatentApplication Publication No. 2010/0113303; PCT Publication No. WO2007/027935; U.S. Pat. No. 7,666,817; U.S. Patent ApplicationPublication No. 20100173349, and US Patent Application Publication No.20130123141, each of which is hereby incorporated by reference in itsentirety.

The CPs and DPs described herein provide unexpected advantages overthese and other previous systems. For example, the systems providedherein allow for the display of larger polypeptides, includingpolypeptides that are at least about 200 amino acids in length. Thedisplay proteins and systems provided herein exhibit increasedflexibility of the N- and C-termini linkers, have truncatedextracellular loops, particularly loop 3 of the transmembrane protein,and are able to display large, intact and functional molecules such asscFv and other antibody fragments at either the C- or N-termini. In someembodiments antibody fragments are displayed at both the N- andC-termini of a CP; for example, a light chain comprising a variablelight (VL) domain and a constant light (CL) domain could be displayed onone terminus and a heavy chain comprising a variable heavy (VH) domainand a constant heavy (CH) domain (e.g., CHO could be expressed on theother terminus in such a conformation as to allow the heavy chain andthe light chain to bind to each other. In some embodiments a receptorand its ligand can be displayed at the N- and C-termini, or C- andN-termini, respectively; such a display system could be used to screenfor inhibitors of receptor-ligand interactions. Display on the systemsprovided herein can be enhanced through E. coli strain selection andculture conditions.

The display peptides and systems described herein provide a novelscaffold for protein engineering for use in antibody affinity maturationand other protein engineering goals with the potential for rapidscreening in a native-like format of activatable antibodies andproteins, such as by way of non-limiting example, those described in PCTPublication Nos. WO 2009/025846, WO 2010/081173; and WO 2010/096838,each of which is herein incorporated by reference in their entirety. Thedisplay peptides and systems described herein can also be used to screenfor substrates and/or masks to be used in activatable antibodies in thepresence of the antibody.

In some embodiments, the systems provided herein include a circularlypermuted transmembrane bacterial CP, such as for example, an E. colitransmembrane protein, in which the N- and C-termini of the expressed,circularly permuted CP are located outside the outer membrane of areplicable biological entity. Previous systems have used a circularlypermuted variant OmpX transmembrane protein, which is shown in FIG. 6A.With N- and C-termini outside the outer membrane by design, peptidedisplay has been possible, enabling sorting of peptide libraries withFACS. (See e.g., Rice et al, Protein Sci. 2006).

The CPs and/or DPs and methods of using these CPs and/or DPs providedherein utilize a different circularly permuted, modified transmembranebacterial protein for polypeptide display, one that is designed toprovide improved display characteristics and increased resistance toprotease degradation. These modified transmembrane proteins arecollectively referred to herein as CYTX carrier proteins (CYTX-CPs). Insome embodiments, the CYTX-CP includes at least the amino acid sequenceof SEQ ID NO: 1. In some embodiments, the CYTX-CP includes at least theamino acid sequence of SEQ ID NO: 56. In some embodiments, the CYTX-CPincludes at least the amino acid sequence of SEQ ID NO: 57.

Display platforms that use these CYTX-CPs to present display moieties onthe outer surface of the CYTX-CP are called “CYTX-DP” (CYTX DisplayPlatforms). CYTX-DPs that are used to identify peptide substratesequences for a given protease or other enzyme are called “CYTX-DP-S” or“CYTX-DP-Substrate” platforms. No structural arrangement is intended bythe order of abbreviations used herein. CYTX-DPs that are used todisplay masking moieties, are referred to herein as “CYTX-DP-MM” or“CYTX-DP-Mask” or “CYTX-DP-Masking Moiety” platforms. CYTX-DPs that areused to display antibodies, antibody fragments and other immunologicalpolypeptides are referred to herein as “CYTX-DP-AB”or “CYTX-DP-Antibody”platforms. CYTX-DPs that are used to display activatable antibodies thatinclude a masking moiety and a substrate are referred to herein as“CYTX-DP-Activatable Antibody” platforms and can display the activatableantibody either as MM-S-AB or AB-S-MM (i.e., they are “CYTX-DP-MM-S-AB”or “CYTX-DP-AB-S-MM” platforms. These platforms are particularly usefulas they allow the selection of masks or substrates in the context of theantibody or antibody binding fragment thereof. In some embodiments, alibrary of CYTX-DP-Activatable Antibodies varies the MM sequences toallow for the selection of a MM while keeping the S and AB constant. Insome embodiments, a library of CYTX-DP Activatable Antibodies varies theS sequences to allow for the selection of a S while keeping the MM andAB constant.

In some embodiments, the displayed moiety (DM) is a polypeptide ofgreater than 25 amino acids, greater than 50 amino acids, greater than75 amino acids, greater than 100 amino acids, greater than 125 aminoacids, greater than 150 amino acids, greater than 175 amino acids,greater than 200 amino acids, greater than 225 amino acids, greater than250 amino acids, greater than 275 amino acids, greater than 300 aminoacids long, greater than 350 amino acids long, greater than 400 aminoacids long, or greater than 450 amino acids long. In some embodiments,the DM is a polypeptide of no more than 8 amino acids, of no more than10 amino acids, of no more than 15 amino acids, of no more than 20 aminoacids, of no more than 25 amino acids, of no more than 30 amino acids,of no more than 35 amino acids, or of no more than 40 amino acids. Forexample, in some embodiments, the displayed polypeptide includes anactive scFv or other antibody fragment, such as a light chain variabledomain, a heavy chain variable domain, one or more variable domains withor without one or more constant domains, or combinations of antibodydomains wherein one domain may be displayed on one part of the DP andanother domain may be displayed on another part of the DP.

The CPs and/or DPs provided herein are useful for screening or otherwiseanalyzing samples from a variety of environments, including complexmixtures with high protease activity and other protease-richenvironments such as tumor sites, synovial fluid, tissue extracts,conditioned media from protease-expressing cells (such as conditionedmedia from tumor cells), sera, or venoms.

In some embodiments, the present invention provides a carrierpolypeptide and a displayed moiety or an expression vector capable ofexpressing and displaying the CP and DM on an outer surface of areplicable biological entity within an extracellular loop of the CP suchthat the DM is capable of interacting with a given ligand. In someembodiments, the carrier protein is a CYTX-CP.

In some embodiments, an extracellular loop of the carrier protein isopened, resulting in an N-terminus exposed on the outer surface, aC-terminus exposed on the outer surface, or both. In some embodiments,the native C-terminus and the native N-terminus are fused together via apeptide linker. In some embodiments, the N-terminus and the C-terminusexposed to the outer surface are accessible by a ligand. In someembodiments, the C-terminus of the DM is fused to the N-terminus of theCP. In some embodiments, the N-terminus of the DM is fused to theC-terminus of the CP. In some embodiments, the carrier CP is a CYTX-CP.

In some embodiments, the replicable biological entity is a bacterialcell, a yeast cell or a mammalian cell. In some embodiments, thereplicable biological entity is a bacterial cell. In some embodiments,the bacterial cell is Escherichia coli, Shigella sonnei, Shigelladysenteriae, Shigella flexneri, Salmonella typhii, Salmonellatyphimurium, Salmonella enterica, Enterobacter aerogenes, Serratiamarcescens, Yersinia pestis, Bacillus cereus, Bacillus subtilis, orKlebsiella pneumoniae.

In some embodiments, the expression vector further comprises a low copyorigin of replication, such as a p15A origin of replication.

In some embodiments, the expression vector further comprises abacteriocidal antibiotic resistance protein encoding gene. In someembodiments, the bacteriocidal antibiotic resistance protein encodinggene encodes chloramphenicol acetyltransferase, beta-lactamase, or aprotein that renders a bacterium resistant to ampicillin, penicillin,tetracycline, or any other antibiotic known to those skilled in the art.

In some embodiments, the expression vector further comprises at leastone Sfi1 endonuclease restriction enzyme site.

In some embodiments, the expression vector further comprises anarabinose araBAD E. coli operon promoter. In some embodiments,expression is induced with the addition of L-arabinose and stopped bythe removal of arabinose and the addition of glucose.

In some embodiments, the present invention provides a host cell thatcomprises an expression vector as provided herein.

In some embodiments, the present invention provides a method of making apolypeptide display library that comprises creating a plurality of DPsand/or expression vectors capable of expressing a plurality of DPsdescribed herein and inducing expression.

In some embodiments, the present invention provides a polypeptide DMexpressed on the outer surface of a replicable biological entity byinducing expression of an expression vector described herein. In someembodiments, the polypeptide DM is expressed in the second extracellularloop of a CYTX-CP.

In some embodiments, the present invention provides an assay method fordetecting, monitoring, or measuring a given ligand in a sample thatcomprises inducing an expression vector described herein to express thepolypeptide DM and then contacting the polypeptide DM with the sampleand observing whether the polypeptide DM interacts with the ligand.

In some embodiments, the carrier polypeptide comprises the amino acidsequence of SEQ ID NO: 1 or an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 1.

In some embodiments, the carrier polypeptide comprises the amino acidsequence of SEQ ID NO: 56 or an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 56.

In some embodiments, the carrier polypeptide comprises the amino acidsequence of SEQ ID NO: 57 or an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 57.

In some embodiments, the carrier polypeptide comprises the amino acidsequence of SEQ ID NO: 4, which contains an EagI restriction site on theC-terminus prior to the histidine tag, or an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 4.

In some embodiments, the carrier polypeptide comprises the amino acidsequence shown below in SEQ ID NO: 58, which contains a NotI restrictionsite on the C-terminus prior to the histidine tag, or an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 58:

(SEQ ID NO: 58) GQSGQEYMPMEGGSGQSGQGSGSNSGSSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGHHHHHHHH*

In some embodiments, the display platform comprises the amino acidsequence of SEQ ID NO: 81 or an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 81.

In some embodiments, the display platform comprises the amino acidsequence of SEQ ID NO: 82 or an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 82.

The invention also provides nucleic acid molecules encoding the carrierpolypeptides disclosed herein, as well as nucleic acid moleculesencoding polypeptides that incorporate the CPs disclosed herein, suchas, for example, the CYTX-DPs disclosed herein, including the CYTX-DPsfor Substrate Selection (S-CPs). The invention also provides vectorsthat include these nucleic acids. The CPs described herein andpolypeptides that incorporate the CPs disclosed herein are produced byculturing a cell under conditions that lead to expression of the CP orpolypeptide that incorporates the CP, wherein the cell includes thesenucleic acid molecules and/or vectors.

In some embodiments, the nucleic acid molecule encodes a polypeptidethat comprises the amino acid sequence of SEQ ID NO: 1 or a nucleotidesequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence that encodes a polypeptide thatcomprises the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the nucleic acid molecule encodes a polypeptidethat comprises the amino acid sequence of SEQ ID NO: 56 or a nucleotidesequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence that encodes a polypeptide thatcomprises the amino acid sequence of SEQ ID NO: 56.

In some embodiments, the nucleic acid molecule encodes a polypeptidethat comprises the amino acid sequence of SEQ ID NO: 57 or a nucleotidesequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence that encodes a polypeptide thatcomprises the amino acid sequence of SEQ ID NO: 57.

In some embodiments, the nucleic acid molecule encodes a polypeptidethat comprises the amino acid sequence of SEQ ID NO: 4 or a nucleotidesequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence that encodes a polypeptide thatcomprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the carrier polypeptide is encoded by a nucleicacid molecule that comprises the nucleotide sequence of SEQ ID NO: 5 ora nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO:5.

The invention also provides methods of CPs described herein andpolypeptides that incorporate the CPs disclosed herein. In oneembodiment, the method includes the steps of (a) culturing a cell thatincludes a nucleic acid construct that encodes the CP or a polypeptidethat incorporates the CP under conditions that lead to expression of theCP or the polypeptide that incorporates the CP; and (b) recovering theCP or the polypeptide that incorporates the CP.

The present invention provides a method for screening a library of cellsor other RBE presenting DPs, e.g., CYTX-DPs, to identify a peptidesubstrate for an enzyme, by contacting an enzyme with a cell libraryenriched for expression of peptide display scaffolds, wherein eachpeptide display scaffold includes a CP, e.g., a CYTX-CP, a candidatepeptide (DM) (in this embodiment, a substrate (S)) and a detectablemoiety (T), wherein the cells of the cell library exhibit a T signalprior to contacting with the enzyme; and detecting the presence orabsence of a T signal, wherein a decrease in the T signal in thepresence of the enzyme as compared to the absence of the enzymeindicates that at least one cell of the cell library expresses acandidate peptide that is a substrate for the enzyme. In someembodiments, the substrate (S) is in an activatable antibody context;i.e., the DM comprises MM-S-AB or AB-S-MM.

The present invention provides a method for screening a library of cellsor other RBE presenting DPs, e.g., CYTX-DPs, to identify an antibody orantigen-binding fragment thereof (AB), by contacting a target with acell library enriched for expression of peptide display scaffolds,wherein each peptide display scaffold includes a CP, e.g., a CYTX-CP anda candidate polypeptide (DM) (in this embodiment, an AB), and detectingthe level of binding between the target and the candidate polypeptides.The level of binding can be detecting using suitable means.

The present invention provides a method for screening a library of cellsor other RBE presenting DPs, e.g., CYTX-DPs, to identify a maskingmoiety (MM), e.g., a peptide that reduces the ability of an antibody orantibody fragment (AB) to bind a target in an activatable antibodycontext, by contacting a target with a cell library enriched forexpression of peptide display scaffolds, wherein each peptide displayscaffold includes a CP, e.g., a CYTX-CP, a candidate peptide (DM) (inthis embodiment, a peptide MM), and an antibody or antigen-bindingfragment thereof (AB) that is specific for the target, and detecting thelevel of binding between the target and the AB. The level of binding canbe detecting using suitable means. At least one of cell of the celllibrary expresses a candidate peptide that is a MM, when the candidatepeptide reduces or otherwise inhibits the ability of the AB to bind tothe target.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed. The accompanying drawings are included to provide a furtherunderstanding of the invention and are incorporated in and constitutepart of this specification, illustrate several embodiments of theinvention and together with the description serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIG. 1 shows a schematic method of screening using protease-resistantCYTX-DP. Panel A shows an initial enrichment step where cells arescreened for those that properly display the candidate substratepeptides (examples of DMs) in the display scaffolds. A detectable moietyis added to a culture of cells and only those cells that display thereporter substrates on the surface of the cell as fusions to theN-terminus of CYTX-DP. Panel B shows a detection step where the enrichedcells are contacted with an enzyme and substrate cleavage is detected byflow cytometry. Substrate libraries are screened by depleting thelibrary pool of clones that do not display a peptide and then enrichingclones with hydrolyzed substrates.

FIG. 2 shows a schematic diagram of a dual color (e.g., fluorescentsignals at two different wavelengths) peptide display screening method.In the absence of a cleavage, typically both a first fluorescent signalat a first wavelength (e.g., Alexa 488, depicted herein as light grey)at the C-terminus of the diagramed scaffold and a second fluorescentsignal at a second wavelength (e.g., PE, depicted herein as dark grey)at the N-terminus are detected. When cleavage occurs, only theC-terminal fluorescent signal (e.g., Alexa 488) is detected, since theN-terminal fluorescent signal (e.g., PE) is removed when the enzymecleaves the substrate.

FIG. 3 shows schematics of exemplary peptide display scaffolds. Panel Ashows an exemplary peptide display scaffold including a transmembraneprotein (CYTX-CP), an N-terminal domain including the DM, a candidatepeptide (DM), and a first detectable moiety (T1), and a C-terminaldomain including a second detectable moiety (T2). Panel B shows anexemplary peptide display scaffold including a transmembrane protein(CYTX-CP), a C-terminal domain including the DM, a candidate peptide(DM), and a first detectable moiety (T1), and an N-terminal domainincluding a second detectable moiety (T2). Panels C and D show exemplarypeptide display scaffolds including a transmembrane protein (CYTX-CP),an N-terminal domain including the first DM, a candidate inhibitorpeptide (C1), the second DM, a substrate for the enzyme (S) and a firstdetectable moiety (T1), and a C-terminal domain including a seconddetectable moiety (T2). Panels E and F show exemplary peptide displayscaffolds including a transmembrane protein (CYTX-CP), a C-terminaldomain including the first DM, a candidate inhibitor peptide (Ci), thesecond DM, a substrate for the enzyme (S) and a first detectable moiety(T1), and an N-terminal domain including a second detectable moiety(T2).

FIG. 4 shows schematics of exemplary peptide display scaffolds. Panels Aand B show exemplary peptide display scaffolds including a transmembraneprotein (CYTX-CP), an N-terminal domain including the first DM, acandidate substrate peptide (Cs), the second DM, an allosteric regulatorfor the enzyme (A) and a first detectable moiety (T1), and a C-terminaldomain including a second detectable moiety (T2). Panels C and D showexemplary peptide display scaffolds including a transmembrane protein(CYTX-CP), a C-terminal domain including the first DM, a candidatesubstrate peptide (Cs), the second DM, an allosteric regulator for theenzyme (A), and a first detectable moiety (T1), and an N-terminal domainincluding a second detectable moiety (T2).

FIG. 5 shows schematics of exemplary peptide display scaffolds. Panel Ashows an exemplary peptide display scaffold including a transmembraneprotein (CYTX-CP), an N-terminal domain including the first DM, a firstcandidate peptide (C1), and a first detectable moiety (T1), and aC-terminal domain including the second DM, a second candidate peptide(C2), and a second detectable moiety (T2). Panel B shows an exemplarypeptide display scaffold including a transmembrane protein (CYTX-CP), aC-terminal domain including the first DM, a first candidate peptide(C1), and a first detectable moiety (T1), and an N-terminal domainincluding the second DM, a second candidate peptide (C2), and a seconddetectable moiety (T2). Panel C shows an exemplary peptide displayscaffold including a transmembrane protein (CYTX-CP), an N-terminaldomain including the first DM, an allosteric regulator (A) and a firstdetectable moiety (T1), and a C-terminal domain including the second DM,a candidate substrate (Cs) and a second detectable moiety (T2). Panel Dshows an exemplary peptide display scaffold including a transmembraneprotein (CYTX-CP), a C-terminal domain including the first DM, anallosteric regulator (A), and a first detectable moiety (T1), and anN-terminal domain including the second DM, a candidate substrate (Cs),and a second detectable moiety (T2). Panel E shows an exemplary peptidedisplay scaffold including a transmembrane protein (CYTX-CP), anN-terminal domain including the first DM, a known substrate (S), and afirst detectable moiety (T1), and a C-terminal domain including thesecond DM, a candidate inhibitor (C3) and a second detectable moiety(T2). Panel F shows an exemplary peptide display scaffold including atransmembrane protein (CYTX-CP), a C-terminal domain including the firstDM, a known substrate (S), and a first detectable moiety (T1), and anN-terminal domain including the second DM, a candidate inhibitor (C3)and a second detectable moiety (T2).

FIG. 6A is an illustration depicting the native E. coli OmpXtransmembrane protein in both three-dimensional representation (leftstructure) and two dimensional representation (middle structure), and acircularly permuted version of the OmpX transmembrane protein (rightstructure). FIG. 6B is an illustration depicting one embodiment of aprotease-resistant, circularly permuted variant of the OmpX polypeptide,referred to herein as “CYTX-CP,” in which loop 3 is shorter andpotential protease cleavage sites in loops 2 and 3 have been replacedwith a flexible glycine-serine based peptide sequence. FIG. 6C is anillustration depicting one embodiment of a protease-resistant displayscaffold, referred to herein as “CYTX-DP”, in which the N-terminus ofthe CYTX-CP is operably linked to the C-terminus of the DM, i.e., arandom candidate peptide library that, in turn, is operably linked tothe sequence EYMPMEGGSG (SEQ ID NO: 31) and, and the C-terminus of theCYTX-CP is operably linked to a histidine tag to produce a displayscaffold that is more compact, more flexible and more resistant toproteases that previous display scaffolds.

FIG. 7 depicts two views of a 3-dimensional X-ray crystallographystructure (grey) of OmpX superimposed on a 3-dimensional NMRspectroscopy structure (black) of OmpX. Loops 2 and 3 are more flexiblein the NMR structure than in the x-ray crystal structure; the NMRstructure also reveals that the beta-strands are more dynamic andtherefore represent potential sources of protease liability.

FIG. 8 is a graph and an illustration depicting the screening of variousaspects of previous cellular libraries of peptide display systems.Truncating loop 3 of circularly permuted OmpX and replacing theC-terminal tag with an 8×HIS tag improved the display characteristics,while loop 2 mutations decreased overall polypeptide display.

FIG. 9 is an illustration comparing previous display systems with oneembodiment of the CYTX-DP display systems provided herein in which thestructures in the tags, linkers, and transmembrane region have beenmodified: The CYTX-CP transmembrane region is compact, the linkers haveshifted, and the N- and C-terminal tags are different. The signalpeptides (ss) are the same, and the substrate site (example of a DM)remains flexible with new flexible restriction sites (unlabeled).

FIG. 10 is a DNA sequence alignment between the CYTX-DP display system(line 1 (SEQ ID NO: 84)) and the CLiPS display system (line 2 (SEQ IDNO: 85)) starting with N-terminal Tag through Stop codon. Between them,there is 25.3% difference and 74.7% identity.

FIG. 11 is an amino acid sequence alignment between the CYTX-DP displaysystem (line 1 (SEQ ID NO: 86)) and CLiPS (line 2 (SEQ ID NO: 87))starting with N-terminal Tag through Stop codon. Between them, there is28.8% difference and 71.2% identity.

FIG. 12 is a schematic diagram of one embodiment of the CYTX-DP displaysystem for display of antibody fragments (scFv) and proteins. A scFv orprotein can be expressed at either the N-terminal or C-terminal end ofthe display platform with or without epitope tags. It is to beappreciated that the mature form of these display platforms lacks thesignal peptide.

FIG. 13 is a graph demonstrating that the observed second order rateconstant (k_(cat)/K_(M), depicted here as kcat/Km) for each substratewas comparable across both the CLiPS and CYTX-DP platforms.

FIGS. 14A and 14B are a series of graphs depicting the N-terminusstability (14A) and C-terminus stability for the CLiPS and CYTX-DPplatforms. FIG. 14A demonstrates that the CYTX-DP platform is 52-foldmore stable than the CLiPS platform, using a non-linear regression fitto determine an EC₅₀. FIG. 14B demonstrates that the CYTX-DP platform is21-fold more stable than the CLiPS platform, using a non-linearregression fit to determine an EC₅₀. EC₅₀=the concentration of proteaserequired to reduce signal by 50%.

FIG. 15 is a graph depicting that the N-terminal affinity tag of theCYTX-DP platform shows increased resistance over CLiPS in the presenceof synovial fluid during a 1 hr incubation at 37° C. Conversion % refersto percent cleavage of the platform by synovial fluid.

FIG. 16 is a graph and an illustration depicting the expression of scFvat the C-terminus of a CYTX-CP in the CYTX-DP platform. Either an F5scFv (shown below as SEQ ID NO: 44) or an OKT3 scFv (shown below as SEQID NO: 45) was expressed fused to the C-terminus of CYTX-CP based uponfluorescence labeling of all cells. Measurement of both N-terminal andC-terminal tags indicated that arabinose-induced cells expressed from2.8- to 9-fold more CYTX-DP-scFvFS-Cterm or CYTX-DP-scFvOKT3-Cterm thanun-induced cells when labeled with the tags at either the N- orC-terminus.

FIG. 17 is a graph and an illustration depicting the expression of scFvat either the N-terminus or the C-terminus of a CYTX-CP in the CYTX-DPplatform. Two different scFvs, F5 and OKT3, were each displayed ateither the N- or C-termini, as determined by measurement of N-terminaltags in arabinose-induced and un-induced cells transformed with vectorsencoding a CYTX-DP-scFvFS-Nterm, CYTX-DP-scFvFS-Cterm,CYTX-DP-scFvOKT3-Nterm, or CYTX-DP-scFvOKT3-Cterm display platform.

FIG. 18 is a series of plots demonstrating that improved expression onthe CYTX-DP platform corresponds to improved antigen binding. Displayplatforms comprising either scFv F5 (CYTX-DP-scFvFS-Nterm) oranti-CTLA-4 clone 2 antibody (CYTX-DP-antiCTLA4-Nterm) were expressed ineither E. coli DH-10β or E. coli C41(DE3), at either 28° C. or 37° C.,as indicated in FIG. 18. Panels A-D depict expression by the varioussystems. Panels E-H depict the ability of the systems to bind CTLA-4antigen. When antiCTLA-4 is expressed at >10% (panel D), the antibodywas able to bind to CTLA-4 antigen at 2.5% (panel H). Expression of theanti-gp130 antibody scFv in C43(DE3) E. coli. Panel I shows N-terminallabeling with anti-EE epitope tag antibody conjugated with Alex488. Sixpercent of the population is expressing the anti-EE epitope tag. Panel Jshows that the anti-gp130 scFv expressing bacteria bind biotinylatedsoluble, human gp130 and are labeled with secondary streptavidin-PE(SAPE) at 1/50 dilution. 2.7% of the population binds soluble gp130.

FIG. 19 is a series of plots comparing scFv expression in the CYTX-DPplatform and the CLiPS platform. Expression of F5 scFv and anti-CTLA-4antibody was significantly more robust in E. coli strains transformedwith CYTX-DP platforms (CYTX-DP-scFvFS-Nterm or CYTX-DP-antiCTLA4-Nterm,respectively) than in E. coli strains C41(DE3) or C43 (DE3) transformedwith CLiPS platforms encoding F5 scFv or anti-CTLA-4.

FIG. 20 is an illustration depicting the amino acid sequence (SEQ ID NO:25) of a CLiPS platform referred to herein as eCLiPS3.0-NSUB_SP.eCLiPS3.0-NSUB_SP includes the following elements: Signal Peptide (SEQID NO: 6)—Linker L1 (SEQ ID NO: 7)—N-terminal SA-tag (SEQ ID NO:14)—Linker L2 (SEQ ID NO: 9)—Linker L3 (SEQ ID NO: 15)—CLiPS3.0transmembrane platform (SEQ ID NO: 16)—Linker L4 (SEQ ID NO:17)—C-terminal MONA tag (SEQ ID NO: 18).

FIG. 21 is an illustration depicting the amino acid sequence (SEQ ID NO:26) of a CLiPS platform referred to herein as eCLiPS3.0-1203_SP.eCLiPS3.0-1203_SP includes the following elements: Signal Peptide (SEQID NO: 6)—Linker L1 (SEQ ID NO: 7)—N-terminal SA-tag (SEQ ID NO:14)—Linker L2 (SEQ ID NO: 9)—1203 Substrate (SEQ ID NO: 19)—Linker L3(SEQ ID NO: 20)—CLiPS3.0 transmembrane platform (SEQ ID NO: 16)—LinkerL4 (SEQ ID NO: 17)—C-terminal MONA tag (SEQ ID NO: 18).

FIG. 22 is an illustration depicting the amino acid sequence (SEQ ID NO:27) of a CLiPS platform referred to herein as eCLiPS3.0-1204_SP.eCLiPS3.0-1204_SP includes the following elements: Signal Peptide (SEQID NO: 6)—Linker L1 (SEQ ID NO: 7)—N-terminal SA-tag (SEQ ID NO:14)—Linker L2 (SEQ ID NO: 9)—1204 Substrate (SEQ ID NO: 21)—Linker L3(SEQ ID NO: 20)—CLiPS3.0 transmembrane platform (SEQ ID NO: 16)—LinkerL4 (SEQ ID NO: 17)—C-terminal MONA tag (SEQ ID NO: 18).

FIG. 23 is an illustration depicting the amino acid sequence (SEQ ID NO:28) of one embodiment of a display platform referred to herein asCYTX-DP-NSUB_SP. The CYTX-DP-NSUB_SP display platform includes thefollowing elements: Signal Peptide (SEQ ID NO: 6)—Linker L1 (SEQ ID NO:7)—N-terminal EE-tag (SEQ ID NO: 8)—Linker L2 (SEQ ID NO: 22)—NSUBsequence (SEQ ID NO: 23)—Linker L3 (SEQ ID NO: 24)—CYTX CP transmembraneportion (SEQ ID NO: 1)—Linker L4 (SEQ ID NO: 12)—C-terminal His tag (SEQID NO: 13).

FIG. 24 is an illustration depicting the amino acid sequence (SEQ ID NO:29) of one embodiment of a display platform referred to herein asCYTX-DP-1203_SP. The CYTX-DP-1203 SP display platform includes thefollowing elements: Signal Peptide (SEQ ID NO: 6)—Linker L1 (SEQ ID NO:7)—N-terminal EE-tag (SEQ ID NO: 8)—Linker L2 (SEQ ID NO: 22)—1203Substrate (SEQ ID NO: 19)—Linker L3 (SEQ ID NO: 24)—CYTX CPtransmembrane portion (SEQ ID NO: 1)—Linker L4 (SEQ ID NO: 12)—C-terminal His tag (SEQ ID NO: 13).

FIG. 25 is an illustration depicting the amino acid sequence (SEQ ID NO:30) of one embodiment of a display platform referred to herein asCYTX-DP-1204_SP. The CYTX-DP-1204 SP platform includes the followingelements: Signal Peptide (SEQ ID NO: 6)—Linker L1 (SEQ ID NO:7)—N-terminal EE-tag (SEQ ID NO: 8)—Linker L2 (SEQ ID NO: 22)-1204Substrate (SEQ ID NO: 21)—Linker L3 (SEQ ID NO: 24)—CYTX CPtransmembrane portion (SEQ ID NO: 1)—Linker L4 (SEQ ID NO:12)—C-terminal His tag (SEQ ID NO: 13).

FIG. 26 is an illustration depicting the amino acid sequence (SEQ ID NO:47) of a CLiPS platform referred to herein as eCLiPS3.0-NSUB.eCLiPS3.0-NSUB is the same as eCLiPS3.0-NSUB_SP except thateCLiPS3.0-NSUB lacks a signal peptide.

FIG. 27 is an illustration depicting the amino acid sequence (SEQ ID NO:48) of a CLiPS platform referred to herein as eCLiPS3.0-1203.eCLiPS3.0-1203 is the same as eCLiPS3.0-1203_SP except thateCLiPS3.0-1203 lacks a signal peptide.

FIG. 28 is an illustration depicting the amino acid sequence (SEQ ID NO:49) of a CLiPS platform referred to herein as eCLiPS3.0-1204.eCLiPS3.0-1204 is the same as eCLiPS3.0-1204_SP except thateCLiPS3.0-1204 lacks a signal peptide.

FIG. 29 is an illustration depicting the amino acid sequence (SEQ ID NO:50) of one embodiment of a display platform referred to herein asCYTX-DP-NSUB. The CYTX-DP-NSUB display platform is the same as theCYTX-DP-NSUB_SP display platform except that CYTX-DP-NSUB lacks a signalpeptide.

FIG. 30 is an illustration depicting the amino acid sequence (SEQ ID NO:51) of one embodiment of a display platform referred to herein asCYTX-DP-1203. The CYTX-DP-1203 display platform is the same as theCYTX-DP-1203_SP display platform except that CYTX-DP-1203 lacks a signalpeptide.

FIG. 31 is an illustration depicting the amino acid sequence (SEQ ID NO:52) of one embodiment of a display platform referred to herein asCYTX-DP-1204. The CYTX-DP-1204 display platform is the same as theCYTX-DP-1204_SP display platform except that CYTX-DP-1204 lacks a signalpeptide.

FIGS. 32A-32D are a series of graphs depicting various populationsanalyzed by flow cytometry. FIG. 32A depicts an initial spiked sample at1:100 (ss1204:ssNSUB) with label only. FIG. 32B depicts an initialspiked sample at 1:100 (ss1204:ssNSUB) cleaved with 100 nM uPA thenlabeled. FIG. 32C depicts a post-sort sample with label only. FIG. 32Ddepicts a post-sort sample (ss1204:ssNSUB) cleaved with 100 nM uPA, thenlabeled.

FIG. 33 is a graph depicting the percentage of cells in the P3 gate.

FIG. 34 is a series of graphs depicting the enrichment of MT-SP1substrates.

FIG. 35 is a series of graphs depicting positional analysis of MT-SP1substrates using the display platforms (DPs) of the disclosure versusphage display.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to display platforms in which a surfacelocalized polypeptide, referred to herein to as a carrier protein (CP),is efficiently expressed on the outer surface of a replicable biologicalentity, where the carrier protein, also referred to herein as carrierpolypeptide, displays one or more given polypeptide(s) or othermolecule(s), a “displayed moiety” (DM) polypeptide to produce a displayplatform (DP).

The purpose of “cell surface display” systems is to present polypeptideson living cells to extracellular targets of any size and molecularcomposition. The application of bacterial display technology to a broadrange of protein engineering applications, however, has been hindered bythe absence of robust, validated display scaffolds. The presentinvention is based on the discovery of circularly permuted transmembranebacterial polypeptides with enhanced properties such as increasedresistance to protease degradation and improved display of largerpolypeptides, for use in bacterial display. Larger polypeptides includenot just longer amino acid sequences, but also polypeptides having alarger molecular weight.

As described in the Examples provided herein, semi-rational design anddirected evolution were used to create circularly permuted outermembrane protein variants also presenting both the N- and C-termini, butshowing significantly enhanced display of a diverse group of peptides,microproteins, and repeat proteins compared to previous display systemsusing circularly permuted OmpX display polypeptides including thosedescribed in PCT Publication No. WO 2005/047461; U.S. Pat. No.7,256,038; U.S. Pat. No. 7,612,019; U.S. Patent Application PublicationNo. 2010/0113303; PCT Publication No. WO 2007/027935; U.S. Pat. No.7,666,817; U.S. Patent Application Publication No. 20100173349, each ofwhich is hereby incorporated by reference in its entirety.

In particular, the new circularly permuted transmembrane bacterialprotein, referred to herein as “CYTX-CP,” has been designed to removepotential protease cleavage sites within the exposed extracellular loopsof the native OmpX sequence, within linker sequences separating theepitope tags from the scaffold and within the N- and C-epitope terminaltags. In some embodiments, these potential protease cleavage sites weresubstituted with a flexible peptide sequence comprised predominantly ofglycine and serine residues (i.e., “Gly-Ser linkers”). This approachprovides a potential route to enhance the performance of a variety ofcell surface display scaffolds in presenting displayed moieties (DM).Thus, the methods described herein can be used to make library screensmore efficient and less biased towards peptides that are difficult todisplay. The CYTX-CPs of the invention include at least the amino acidsequence of SEQ ID NO: 1. In some embodiments, the CYTX-CPs of theinvention include at least the amino acid sequence of SEQ ID NO: 56. Insome embodiments, the CYTX-CPs of the invention include at least theamino acid sequence of SEQ ID NO: 57.

The disclosure provides a CYTX-CP comprising any circularly permutedbacterial outer membrane protein (Omp) that comprises one or moreextracellular loops. In some embodiments, the Omp is OmpX. In someembodiments, the Omp is OmpA. In some embodiments, the Omp is OmpG.

In some embodiments, the polypeptide display platforms (DPs) include (i)one or more carrier polypeptides (CPs) having at least a transmembraneportion and N- and C-termini that are exposed when the CP is displayedon the outer membrane of a replicable biological entity, and (ii) adisplayed moiety (DM), such as for example, a polypeptide or otherbiological molecule. Suitable DMs include, by way of non-limitingexample, a substrate sequence (S), e.g., a peptide sequence that iscleaved by one or more proteases; a masking moiety (MM), e.g., a peptidesequence that reduces the ability of an antibody or antibody fragment(AB) to bind a target; an exosite (EX); an allosteric binding site (AS);an antibody or antibody fragment (AB); a receptor (R); a ligand (L); aninhibitor (I); and any combination thereof. In some embodiments, CPshaving only one DM have the general structural arrangement fromN-terminus to C-terminus as follows: DM-CP or CP-DM.

In some embodiments, the DM is a masking moiety (MM), e.g., a peptidesequence that reduces the ability of an antibody or antibody fragment(AB) to bind a target. In some embodiments, the target is a targetselected from those shown below in Table 1.

TABLE 1 Exemplary Targets 1-92-LFA-3 CD117 ERBB3 IGF1R MUC1 TLR4Anti-Lewis-Y CD132 F protein of IL1B Mucin-16 TLR6 (IL-2RG) RSV Apelin JCD133 FAP IL1R Na/K TLR7 receptor ATPase APRIL CD137 FGF-2 IL2Neutrophil elastase TLR8 BAFF CD138 FGF8 IL11 NGF TLR9 C5 CD172A FGFR1IL12 Nicastrin TNFalpha complement C-242 CEACAM5 FGFR2 IL12p40 NotchReceptors TNFR (CEA) CD2 CEACAM6 FGFR3 IL-12R, IL- Notch 1 TRAIL-R1(NCA-90) 12Rbeta1 CD3 CLAUDIN-3 FGFR4 IL13 Notch 2 TRAIL-R2 CD9CLAUDIN-4 Folate receptor IL13R Notch 3 Transferrin CD11a cMet G-CSFIL15 Notch 4 Transferrin receptor CD19 Collagen G-CSFR IL17 NOV TRK-ACD20 Cripto GLUT1 IL18 OSM-R TRK-B CD22 CSFR GLUT4 IL21 PAR2 uPAR CD25CSFR-1 GM-CSF IL23 PDGF-AA VCAM-1 CD28 CTLA-4 GM-CSFR IL23R PDGF-BB VEGFCD30 CTGF GP IIb/IIIa IL27/IL27R PDGFRalpha VEGF-A receptors (wsx1) CD33CXCL10 Gp130 IL29 PDGFRbeta VEGF-B CD40 CXCL13 GPIIB/IIIA IL-31R PD-1VEGF-C CD40L CXCR1 GPNMB IL31/IL31R PD-L1, VEGF-D PD-L2 CD41 CXCR2HER2/neu IL2R Phosphatidyl- VEGFR1 serine CD44 CXCR4 HGF IL4 P1GF VEGFR2CD47 CYR61 hGH IL4R PSCA VEGFR3 CD52 DL44 Hyaluronidase IL6, IL6R PSMAWISP-1 CD56 DLL4 IFNalpha Insulin RAAG12 WISP-2 Receptor CD64 DPP-4IFNbeta Jagged RAGE WISP-3 Ligands CD70 EGFR IFNgamma Jagged 1 SLC44A4Alpha-4 integrin CD80 Endothelin B IgE Jagged 2 Sphingosine Alpha-Vreceptor 1 Phosphate integrin (ETBR) CD86 EpCAM IgE Receptor LIF-RTGFbeta alpha4beta (FceRI) 1 integrin CD95 EPHA2 IGF MRP4 TLR2alpha4beta 7 integrin

In some embodiments, the DM is a substrate sequence (S), e.g., a peptidesequence that is cleaved by one or more proteases. In some embodiments,the protease is a protease selected from those shown in Table 2.

TABLE 2 Exemplary Proteases ADAMS, ADAMTS, e.g. ADAM8 ADAM9 ADAM10ADAM12 ADAM15 ADAM17/TACE ADAMTS1 ADAMTS4 ADAMTS5 Aspartate proteases,e.g., BACE Aspartic cathepsins, e.g., Cathepsin D Cathepsin E Caspases,e.g., Caspase 1 Caspase 2 Caspase 3 Caspase 4 Caspase 5 Caspase 6Caspase 7 Caspase 8 Caspase 9 Caspase 10 Caspase 14 Cysteine cathepsins,e.g., Cathepsin B Cathepsin C Cathepsin K Cathepsin L Cathepsin SCathepsin V/L2 Cathepsin X/Z/P Cysteine proteinases, e.g., CruzipainLegumain KLKs, e.g., KLK4 KLK5 KLK6 KLK7 KLK8 KLK10 KLK11 KLK13 KLK14Metallo proteinases, e.g., Meprin Neprilysin PSMA BMP-1 MMPs, e.g.,MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14MMP-15 MMP-19 MMP-23 MMP-24 MMP-26 MMP-27 Serine proteases, e.g.,activated protein C Cathepsin A Cathepsin G Chymase coagulation factorproteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa) Elastase Granzyme BGuanidinobenzoatase Human Neutrophil Elastase NS3/4A Plasmin PSA tPAThrombin Tryptase uPA Type II Transmembrane Serine Proteases (TTSPs),e.g., DESC1 DPP-4 FAP Hepsin Matriptase-2 MT-SP1/Matriptase TMPRSS2TMPRSS3 TMPRSS4

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a peptide” includes a mixture of two or more suchpeptides, and the like.

As used herein, a “replicable biological entity” refers toself-replicating biological cells, including bacterial, yeast,protozoal, and mammalian cells, and various viruses capable of infectingthese cells known in the art, and the like.

The term “CPX” as used herein refers to a circularly permuted variant ofa bacterial outer membrane protein OmpX (see U.S. Pat. No. 7,256,038,which is herein incorporated by reference in its entirety). The CPXprotein consists of the native OmpX signal sequence, which is cleavedafter translocation; a sequence with an embedded SfiI restriction site(GQSGQ) (SEQ ID NO: 7) after which peptides may be inserted; a flexiblelinking sequence (GGQSGQ) (SEQ ID NO 32); amino acids S54-5 F148 of themature OmpX; a GGSG linker joining the native C- and N-termini of OmpX;and amino acids A1-S53 of the mature OmpX. CPX can be used as a proteinscaffold for bacterial display of peptides and proteins at the surfaceof a bacterial cell. Furthermore, for purposes of the present invention,the term “eCPX” refers to a protein that includes modifications, such asdeletions, additions and substitutions, for example, replacement of thelinker joining the native N- and C-termini of OmpX, substitutions atpositions 165 and 166 (numbered with reference to the sequence of nativeOmpX from Escherichia coli, SEQ ID NO: 1 in PCT Publication No. WO2009/014726), incorporation of alternate restriction sites after whichpolypeptides or peptides may be inserted, or the addition of linkersbetween the N-terminus or C-terminus of eCPX and a DM, so long as theprotein maintains biological activity (i. e., ability to efficientlydisplay polypeptides) (see e.g., PCT Publication No. WO 2009/014726).These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hoststhat produce the proteins or errors due to PCR amplification.

As used herein, the term “CYTX-CP” refers to a circularly permutedvariant of an OmpX protein having one or more of the followingmutations: (i) potential protease cleavage sites on the exposed portionof the OmpX protein (i.e., outside the outer membrane) have beenreplaced, for example, with a glycine-serine based flexible peptidesequence; (ii) one or more epitope tags has been modified to minimizetag proteolysis; (iii) modifications in loop 2 of the OmpX protein;and/or (iv) modifications in loop 3 of the OmpX protein. For example,modification of loop 2 and/or loop 3 can include replacing a portion ofthe loop with a shorter, non-cleavable amino acid sequence. Modificationof loop 2 and/or loop 3 can include replacing existing potentialcleavage sites within the loop with flexible, glycine-serine based aminoacid sequences that are generally non-cleavable by proteases. In someembodiments, a CYTX-CP is derived from an Omp other than OmpX that hasat least one extracellular loop. Examples include, but are not limitedto, circularly permuted OmpA and OmpG proteins.

An advantage of using CP, e.g., CYTX-CP, in bacterial display is thatboth its N- and C-termini are exterior to the cell, which allowspolypeptides to be displayed from either terminus or from both terminisimultaneously. The term CP includes circularly permuted variants ofOmpX from any strain of bacteria, such as Escherichia coli, Shigellasonnei, Shigella dysenteriae, Shigella flexneri, Salmonella typhii,Salmonella typhimurium, Salmonella enterica, Enterobacter aerogenes,Serratia marcescens, Yersinia pestis, Bacillus cereus, Bacillussubtilis, or Klebsiella pneumoniae. The GenBank database containscomplete sequences for OmpX proteins from a variety of bacterialisolates, which could be used to produce CP proteins of the invention.The GenBank database also contains complete sequences for other Ompproteins from a variety of bacterial isolates, which could be used toproduce CP proteins of the invention.

As used herein, the terms “protein”, “polypeptide” and “peptide” areused interchangeably to refer to two or more amino acids linkedtogether.

The terms “polypeptide”, “peptide”, “protein”, and “amino acid sequence”as used herein generally refer to any compound comprising naturallyoccurring or synthetic amino acid polymers or amino acid-like moleculesincluding but not limited to compounds comprising amino and/or iminomolecules. No particular size is implied by use of the term “peptide”,“oligopeptide”, “polypeptide”, or “protein” and these terms are usedinterchangeably. Included within the definition are, for example,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), polypeptides with substitutedlinkages, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring (e.g., synthetic). Thus,synthetic oligopeptides, dimers, multimers (e.g., tandem repeats,multiple antigenic peptide (MAP) forms, linearly-linked peptides),cyclized, branched molecules and the like, are included within thedefinition. The terms also include molecules comprising one or morepeptoids (e.g., N-substituted glycine residues) and other syntheticamino acids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005;5,877,278; and 5,977,301; Nguyen et al. (2000) Chem. Biol. 7(7):463-473;and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 fordescriptions of peptoids). Non-limiting lengths of peptides suitable foruse in the present invention includes peptides of 3 to 5 residues inlength, 6 to 10 residues in length (or any integer therebetween), 11 to20 residues in length (or any integer therebetween), 21 to 75 residuesin length (or any integer therebetween), 75 to 100 (or any integertherebetween), or polypeptides of greater than 100 residues in length.

Typically, polypeptides useful in this invention can have a maximumlength suitable for the intended application. In some embodiments, thepolypeptide is between about 3 and 300 amino acid residues in length. Insome embodiments, the polypeptide is more than 300 amino acid residuesin length. In some embodiments, the polypeptide is more than 350 aminoacid residues in length. In some embodiments, the polypeptide is morethan 400 amino acid residues in length. In some embodiments, thepolypeptide is more than 450 amino acid residues in length. In someembodiments, the DM is a polypeptide of no more than 8 amino acids, ofno more than 10 amino acids, of no more than 15 amino acids, or of nomore than 40 amino acids. Generally, one skilled in art can easilyselect the maximum length in view of the teachings herein. Further,peptides as described herein, for example synthetic peptides, mayinclude additional molecules such as labels or other chemical moieties(e.g., streptavidin conjugated to phycoerythrin, Alexa dye conjugated toanti-T7 tag, Alexa dye conjugated to an anti-EE antibody, Alexa dyeconjugated to an anti-His antibody). Such moieties may further enhanceinteraction of the peptides with a ligand and/or further detection ofpolypeptide display.

Thus, reference to peptides also includes derivatives of the amino acidsequences of the invention including one or more non-naturally occurringamino acid. A first polypeptide is “derived from” a second polypeptideif it is (i) encoded by a first polynucleotide derived from a secondpolynucleotide encoding the second polypeptide, or (ii) displayssequence identity to the second polypeptide as described herein.Sequence (or percent) identity can be determined as described below. Insome embodiments, derivatives exhibit at least about 50% percentidentity, in some embodiments, at least about 80%, and in someembodiments, between about 85% and 99% (or any value therebetween) tothe sequence from which they were derived. Such derivatives can includepost-expression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation, and the like.

Amino acid derivatives can also include modifications to the nativesequence, such as deletions, additions and substitutions (generallyconservative in nature), so long as the polypeptide maintains thedesired activity. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts that produce the proteins or errors due to PCRamplification. Furthermore, modifications may be made that have one ormore of the following effects: increasing efficiency of bacterialdisplay, level of expression, or stability of the polypeptide.Polypeptides described herein can be made recombinantly, synthetically,or in tissue culture.

Polypeptides presented according to the present invention (1) alleviatedisruption of the energetic structural stability of the carrierpolypeptide thus allowing presentation of suitable number of copies ofthe DM exhibiting acceptable viability, (2) are capable of interactingphysically with arbitrary compositions of matter (biological ornon-biological), and (3) exhibit a biological activity (e.g., affinity,specificity, catalysis, assembly etc.) substantially similar to thecorresponding free polypeptide in solution. In other words, thedisplayed polypeptide interacts with or binds a given target molecule ina manner that is substantially similar to that when the polypeptide isin its native environment and not attached to the biological entity.

As used herein, a “fusion protein” refers to the expression product oftwo or more nucleic acid molecules that are not natively expressedtogether as one expression product. For example, a native protein Xcomprising subunit A and subunit B, which are not natively expressedtogether as one expression product, is not a fusion protein. However,recombinant DNA methods known in the art may be used to express subunitsA and B together as one expression product to yield a fusion proteincomprising subunit A fused to subunit B. A fusion protein may compriseamino acid sequences that are heterologous, e.g., not of the sameorigin, not of the same protein family, not functionally similar, andthe like.

The polypeptides expressed and displayed according to the presentinvention may be large polypeptides yet still retain the ability to bindor interact with given ligands in a manner similar to the nativepolypeptide or the polypeptide in solution. As provided herein, theexpression vectors of the present invention use utilize a low copyorigin of replication and a regulatable promoter in order to minimizethe metabolic burden of the biological entity and the clonalrepresentation of the polypeptide library is not affected by growthcompetition during library propagation. The expression vectors of thepresent invention utilize an antibacterial resistance gene to abacteriocidal antibiotic that prevents plasmid loss and outgrowth ofcells resistant to the antibiotic. Additionally, the expression vectorsof the present invention lack a dual system, such as 13-lactamase, whichresults in a smaller expression vector that imposes a smaller burden oncell growth and improves library screening. The expression vectors ofthe present invention also utilize a restriction site that allowsdigestion by a particular enzyme to generate overhangs that cannot reactwith incorrect DNA substrates, e.g., such sites enable directionalcloning. Examples of such restriction sites include by are not limitedto EagI, NotI and SfiI.

As used herein, a “ligand” refers to a molecule(s) that binds to anothermolecule(s), e.g., an antigen binding to an antibody, a hormone orneurotransmitter binding to a receptor, or a substrate or allostericeffector binding to an enzyme and include natural and syntheticbiomolecules, such as proteins, polypeptides, peptides, nucleic acidmolecules, carbohydrates, sugars, lipids, lipoproteins, small molecules,natural and synthetic organic and inorganic materials, syntheticpolymers, and the like.

As used herein, a “receptor” refers to a molecular structure within acell or on the surface characterized by (1) selective binding of aspecific substance and (2) a specific physiologic effect thataccompanies the binding, e.g., membrane receptors for peptide hormones,neurotransmitters, antigens, complement fragments, and immunoglobulinsand nuclear receptors for steroid hormones and include natural andsynthetic biomolecules, such as proteins, polypeptides, peptides,nucleic acid molecules, carbohydrates, sugars, lipids, lipoproteins,small molecules, natural and synthetic organic and inorganic materials,synthetic polymers, and the like.

As used herein, “specifically binds” refers to the character of areceptor that recognizes and interacts with a ligand but does notsubstantially recognize and interact with other molecules in a sampleunder given conditions.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,a-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carboxylic sugar analogs. Examples ofmodifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids”, whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

An “isolated” nucleic acid molecule or polypeptide refers to a nucleicacid molecule or polypeptide that is in an environment that is differentfrom its native environment in which the nucleic acid molecule orpolypeptide naturally occurs. Isolated nucleic acid molecules orpolypeptides includes those having nucleotides or amino acids flankingat least one end that is not native to the given nucleic acid moleculeor polypeptide. For example, a promoter P for a protein X is inserted atthe 5′ end of a protein Y that does not natively have P at its 5′ end.Protein Y is thus considered to be “isolated”.

The term “polynucleotide”, as known in the art, generally refers to anucleic acid molecule. A “polynucleotide” can include both double- andsingle-stranded sequences and refers to, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from viral, prokaryotic oreukaryotic mRNA, genomic RNA and DNA sequences from viral (e.g., RNA andDNA viruses and retroviruses), prokaryotic DNA or eukaryotic (e.g.,mammalian) DNA, and especially synthetic DNA sequences. The term alsocaptures sequences that include any of the known base analogs of DNA andRNA, and includes modifications such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts including polynucleotides encoding CP or a variantthereof. Modifications of polynucleotides may have any number of effectsincluding, for example, facilitating expression/bacterial display of thepolypeptide product at the surface of a host cell.

A polynucleotide can encode a biologically active (e.g., CP or a variantthereof) protein or polypeptide. Depending on the nature of thepolypeptide encoded by the polynucleotide, a polynucleotide can includeas little as 10 nucleotides, e.g., where the polynucleotide encodes alinker, tag or label, or an antigen or epitope for bacterial display.Typically, the polynucleotide encodes peptides of at least 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 oreven more amino acids. The disclosure provides polynucleotides thatencode polypeptides of at least 40, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, or even more aminoacids. In some embodiments, the DM is a polypeptide of no more than 8amino acids, of no more than 10 amino acids, of no more than 15 aminoacids, or of no more than 40 amino acids.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin that, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein,polypeptide, or peptide means a polypeptide produced by expression of arecombinant polynucleotide. In general, the gene of interest is clonedand then expressed in transformed organisms, as described further below.The host organism expresses the foreign gene to produce the proteinunder expression conditions.

A “polynucleotide coding sequence” or a sequence that “encodes” aselected polypeptide, is a nucleic acid molecule that is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vivo when placed under the control of appropriate regulatorysequences (or “control elements”). The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A transcriptiontermination sequence may be located 3′ to the coding sequence. Typical“control elements,” include, but are not limited to, transcriptionregulators, such as promoters, transcription enhancer elements,transcription termination signals, and polyadenylation sequences; andtranslation regulators, such as sequences for optimization of initiationof translation, e.g., Shine-Dalgarno (ribosome binding site) sequences,Kozak sequences (i.e., sequences for the optimization of translation,located, for example, 5′ to the coding sequence), leader sequences(heterologous or native), translation initiation codon (e.g., ATG), andtranslation termination sequences. Promoters can include induciblepromoters (where expression of a polynucleotide sequence operably linkedto the promoter is induced by an analyte, cofactor, regulatory protein,etc.), repressible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), and constitutive promoters.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The terms “label” and “detectable label” refer to a molecule capable ofdetection, including, but not limited to, radioactive isotopes,fluorescers, chemiluminescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, chromophores, dyes, metal ions, metalsols, ligands (e.g., biotin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof that is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels that may be used with the invention include, but are notlimited to phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascadeblue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone,Texas red, luminol, acradimum esters, biotin, green fluorescent protein(GFP), enhanced green fluorescent protein (EGFP), yellow fluorescentprotein (YFP), enhanced yellow fluorescent protein (EYFP), bluefluorescent protein (BFP), red fluorescent protein (RFP), fireflyluciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradishperoxidase, glucose oxidase, alkaline phosphatase, chloramphenicalacetyl transferase, and urease.

The term “derived from” is used herein to identify the original sourceof a molecule but is not meant to limit the method by which the moleculeis made, which can be, for example, by chemical synthesis or recombinantmeans.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of the reference molecule that retain desired activity(e.g., efficient polypeptide display) as described herein. In general,the terms “variant” and “analog” refer to compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions and/or deletions (e.g., in the linker joiningnative N- and C-termini or at positions 165 and 166), relative to thenative molecule, so long as the modifications do not destroy biologicalactivity and that are “substantially homologous” to the referencemolecule as defined below. In general, the amino acid sequences of suchanalogs will have a high degree of sequence homology to the referencesequence, e.g., amino acid sequence homology of more than 50%, generallymore than 60%-70%, even more particularly 80%-85% or more, such as atleast 90%-95% or more, when the two sequences are aligned. Often, theanalogs will include the same number of amino acids but will includesubstitutions, as explained herein. The term “mutein” further includespolypeptides having one or more amino acid-like molecules including butnot limited to compounds comprising only amino and/or imino molecules,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), polypeptides with substitutedlinkages, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring (e.g., synthetic),cyclized, branched molecules and the like. The term also includesmolecules comprising one or more N-substituted glycine residues (a“peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S.Pat. Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem.Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA(1992) 89:9367-9371 for descriptions of peptoids). In some embodiments,the analog or mutein has at least the same polypeptide displayefficiency as the native OmpX molecule. Methods for making polypeptideanalogs and muteins are known in the art and are described furtherbelow.

Analogs generally include substitutions that are conservative in nature,i.e., those substitutions that take place within a family of amino acidsthat are related in their side chains. Specifically, amino acids aregenerally divided into four families: (1) acidic—aspartate andglutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine,cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified as aromatic amino acids. For example,it is reasonably predictable that an isolated replacement of leucinewith isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar conservative replacement of an amino acidwith a structurally related amino acid, will not have a major effect onthe biological activity. For example, the polypeptide of interest mayinclude up to about 5-10 conservative or non-conservative amino acidsubstitutions, or even up to about 15-25 conservative ornon-conservative amino acid substitutions, or any integer between 5-25,so long as the desired function of the molecule remains intact. One ofskill in the art may readily determine regions of the molecule ofinterest that can tolerate change by reference to Hopp/Woods (see e.g.,Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828 (1981)) and Kyte-Doolittleplots (see e.g., J. Mol. Biol. 157:105-132 (1982)), well known in theart.

By “derivative” is intended any suitable modification of the nativepolypeptide of interest, of a fragment of the native polypeptide, or oftheir respective analogs, such as glycosylation, phosphorylation,polymer conjugation (such as with polyethylene glycol), or otheraddition of foreign moieties, so long as the desired biological activityof the native polypeptide is retained. Methods for making polypeptidefragments, analogs, and derivatives are generally available in the art.

By “fragment” is intended a molecule consisting of only a part of theintact full-length sequence and structure. The fragment can include aC-terminal deletion an N-terminal deletion, and/or an internal deletionof the peptide. Active fragments of a particular protein or peptide willgenerally include at least about 5-10 contiguous amino acid residues ofthe full-length molecule, in some embodiments, at least about 15-25contiguous amino acid residues of the full-length molecule, and in someembodiments, at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, or any integer between 5 amino acids andthe full-length sequence, provided that the fragment in question retainsbiological activity, such as ligand-binding activity, as defined herein.

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, in some embodiments, 80%-85%, and insome embodiments, 90-95% of the sample. Techniques for purifyingpolynucleotides and polypeptides of interest are well-known in the artand include, for example, ion-exchange chromatography, affinitychromatography and sedimentation according to density.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two nucleic acid, or two polypeptide sequencesare “substantially homologous” to each other when the sequences exhibitat least about 50%, in some embodiments, at least about 75%, in someembodiments, at least about 80%-85%, in some embodiments, at least about90%, and in some embodiments, at least about 95%-98% sequence identityover a defined length of the molecules. As used herein, substantiallyhomologous also refers to sequences showing complete identity to thespecified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two molecules(the reference sequence and a sequence with unknown % identity to thereference sequence) by aligning the sequences, counting the exact numberof matches between the two aligned sequences, dividing by the length ofthe reference sequence, and multiplying the result by 100. Readilyavailable computer programs can be used to aid in the analysis, such asALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O.Dayhoff ed., 5 Suppl. 3:353-358, National biomedical ResearchFoundation, Washington, D.C., which adapts the local homology algorithmof Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 forpeptide analysis. Programs for determining nucleotide sequence identityare available in the Wisconsin Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.) for example, theBESTFIT, FASTA and GAP programs, which also rely on the Smith andWaterman algorithm. These programs are readily utilized with the defaultparameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above. For example,percent identity of a particular nucleotide sequence to a referencesequence can be determined using the homology algorithm of Smith andWaterman with a default scoring table and a gap penalty of sixnucleotide positions.

Expression Systems Using Protease-Resistant Circularly Permuted OmpXVariants

As provided herein, the carrier polypeptides (CPs), display platforms(DPs), nucleic acids, expression vectors and libraries of the presentinvention incorporate (1) the use of a regulatable expression vectorthat allows on-off control of carrier polypeptide production, (2)efficient restriction sites immediately adjacent to the randomized siteto facilitate high-efficiency cloning, (3) random polypeptides insertedinto non-conserved sites of carrier polypeptide extracellular loops thatefficiently presents a DM to a given ligand, (4) time andtemperature-controlled induction periods to obtain optimal displaylevels that result in higher quality results, (5) the use of a bacterialstrain having a high plasmid transformation efficiency fortransformation, (6) the use of optimized library construction protocolsto construct the largest libraries, (7) the use of multiple-plasmidtransformation to yield a larger number of unique DMs for a given numberof host cells, (8) the use of cell concentration to enable completeprocessing of larger numbers of sequences (e.g., 10¹¹-10¹³), or (9) acombination thereof.

The systems provided herein provide a number of advantages over previousexpression systems. For example, the systems provided herein are moreresistant to protease degradation as compared to prior expressionsystems, including, for example, those described in PCT Publication No.WO 2005/047461, WO 2007/027935 and WO 2009/014726, each of which ishereby incorporated by reference in their entirety. In addition, thesystems provided herein allow for the stable display of a larger numberof polypeptides and other amino acid sequences.

In the systems provided herein, a circularly permuted outer membraneprotein X (OmpX) variant was used as the template for the new displayscaffold. The modifications made in the display and selection scaffoldresults in a protein that is at least 5% smaller, for example, at least10% smaller, at least 15%, at least 20% or at least 25% smaller, andshares less than 100% identity with the OmpX proteins described, forexample, in PCT Publication No. WO 2005/047461, WO 2007/027935 and WO2009/014726, e.g., less than 95%, less than 90%, less than 85%, lessthan 80% identity with the OmpX proteins described, for example, in PCTPublication No. WO 2005/047461, WO 2007/027935 and WO 2009/014726. Thesystems provided herein demonstrate improved cell surface displayallowing for more extensive libraries and improved quality of screening.Previous systems were susceptible to degradation by a variety ofproteases and comprised smaller DMs.

The systems provided herein overcome these problems with previoussystems. The systems provided herein allow for unbiased substratescreening of tissue extracts/biological fluids (positive and negativeselections), the screening process of a wide panel of purified proteases(negative selections) and the screening of larger polypeptide domainlibraries.

The systems provided herein were generated by identifying potentialprotease cleavage sites within the exposed extracellular loops of thepublished OmpX sequence (see e.g., Rice et al., “Bacterial display usingcircularly permuted outer membrane protein OmpX yields high affinitypeptide ligands.” Protein Sci. 2006 15(4):825-36; see also PCTPublication No. WO 2005/047461; PCT Publication No. WO 2009/014726; U.S.Pat. No. 7,256,038; U.S. Pat. No. 7,612,019; U.S. Patent ApplicationPublication No. 2010/0113303; PCT Publication No. WO 2007/027935; U.S.Pat. No. 7,666,817; U.S. Patent Application Publication No. 20100173349,each of which is hereby incorporated by reference in its entirety),within linker sequences separating the epitope tags from the scaffold,within the N- and C-epitope terminal tags, and within otherextracellular regions of the platform. Nucleic acid sequences encodingfor OmpX-variant derived sequences were designed to eliminate potentialprotease cleavage sites by substituting flexible, turned linkerscomprised predominantly of glycine and serine residues (i.e., “Gly-Serlinkers”). The nucleic acid sequences were made by PCR and standardmolecule biology techniques and transformed into bacteria.

A CP polypeptide or protein molecule, as defined above, is asubstrate-resistant circularly permuted variant of a bacterial outermembrane protein derived from bacteria, including, but not limited toEscherichia coli, Shigella sonnei, Shigella dysenteriae, Shigellaflexneri, Salmonella typhii, Salmonella typhimurium, Salmonellaenterica, Enterobacter aerogenes, Serratia marcescens, Yersinia pestis,Bacillus cereus, Bacillus subtilis, or Klebsiella pneumoniae. Themolecule need not be physically derived from the particular isolate inquestion, but may be synthetically or recombinantly produced.

The amino acid sequences of a number of OmpX proteins are known.Representative sequences from bacteria are listed in the National Centerfor Biotechnology Information (NCBI) database. See, for example, NCBIentries: Escherichia coli OmpX, Accession No. P0A917; Serratiamarcescens OmpX, Accession No. AAS78634; Salmonella enterica subsp.enterica serovar Choleraesuis str. SC-B67 ail and ompX homolog,Accession No. YP_219185; Salmonella enterica subsp. enterica serovarTyphi OmpX precursor, Accession No. CAD05280; Enterobacter cloacae OmpX,Accession No. P25253; Yersinia pseudotuberculosis IP 32953 OmpX,Accession No. YP_071052; Yersinia pseudotuberculosis IP 32953 OmpX,Accession No. YP_071052; Shigella flexneri OmpX precursor, Accession No.P0A920; Escherichia coli OmpX precursor, Accession No. P0A918;Escherichia coli OmpX precursor, Accession No. P0A919; Salmonellaenterica subsp. enterica serovar Typhi Ty2 OmpX, Accession No.NP_805818; Shigella flexneri 2a str. 301 OmpX, Accession No. NP_706692;Yersinia pestis KIM OmpX, Accession No. NP_669000; Salmonella entericasubsp. enterica serovar Typhi str. CT18 OmpX, Accession No. NP_455368;Salmonella typhimurium LT2 OmpX, Accession No. NP_459810; Escherichiacoli 0157:H7 str. Sakai OmpX, Accession No. NP_308919; Escherichia coli0157:H7 EDL933 OmpX, Accession No. NP_286578; Shigella flexneri 2a str.2457T OmpX, Accession No. NP_836469; Salmonella enterica subsp. entericaserovar Choleraesuis str. SC-B67 OmpX, Accession No. YP_215816; Yersiniapestis C092 OmpX, Accession No. NP_406040; Yersinia pestis biovarMicrotus str. 91001 OmpX, Accession No. NP_993650; Escherichia coliCFT073 OmpX, Accession No. NP_752830; Salmonella enterica subsp.enterica serovar Paratyphi A str. ATCC 9150 OmpX, Accession No.YP_151143; Erwinia carotovora subsp. atroseptica SCR11043 OmpX,Accession No. YP_050855; Erwinia carotovora subsp. atroseptica SCR11043OmpX, Accession No. YP_050855; Escherichia coli APEC 01 OmpX precursor,Accession No. ABJ00194; Shigella boydii Sb227 OmpX, Accession No.YP_407207; Escherichia coli UTI89 OmpX, Accession No. ABE06304; Yersiniapestis KIM OmpX, Accession No. NP_669349; Yersinia pestis KIM OmpX,Accession No. NP_668646; Escherichia coli 0157:H7 EDL933 OmpX, AccessionNo. AAG55186; Shigella flexneri 2a str. 2457T OmpX, Accession No. 15AAP16275; Escherichia coli APEC 01 OmpX precursor, Accession No.YP_851908; Escherichia coli UTI89 OmpX, Accession No. YP_539835; andShigella sonnei Ss046 OmpX, Accession No. YP_309776; all of whichsequences (as entered by the date of filing of this application) areherein incorporated by reference.

The term “displayed” polypeptide, also referred to herein as a displaymoiety, a displayed moiety or DM, refers to a polypeptide or othermolecule linked to the N- or C-terminus of a carrier polypeptide, e.g.,CYTX-CP, or a variant thereof for display at the surface of a bacterialcell to produce a display platform (DP). In some embodiments, a DM iscapable of interacting physically with arbitrary compositions of matter(biological or non-biological), and exhibits a biological activity(e.g., affinity, specificity, catalysis, assembly etc.) substantiallysimilar to the corresponding free polypeptide in solution. In otherwords, the DM interacts with or binds a given target molecule in amanner that is substantially similar to that when the polypeptide is inits native environment and not attached to the CP, e.g., CYTX-CP, or avariant thereof.

Bacterial display can be used in combination with magnetic-activatedcell sorting (MACS) and/or fluorescence-activated cell sorting (FACS)and/or other affinity-based selection techniques for quantitativelibrary analysis and screening for CYTX-DPs that display polypeptides orpeptides efficiently (see, e.g., Rice et al. (2006) Protein Sci.15:825-836; U.S. Patent Application Publication No. 2005/0196406;Daugherty et al. (2000) J. Immuunol. Methods 243(1-2):211-2716; Georgiou(2000) Adv. Protein Chem. 55:293-315; Daugherty et al. (2000) Proc.Natl. Acad. Sci. U.S.A. 97(5):20293418; Olsen et al. (2003) Methods Mol.Biol. 230:329-342; and Boder et al. (2000) Proc. Natl. Acad. Sci. U.S.A.97(20):10701-10705; herein incorporated by reference in theirentireties). Analysis of the display efficiency of a CYTX-DP isfacilitated by the use of a display system comprising a label (e.g.,phycoerythrin, Alexa dye, fluorescein, YPet, CyPet) that allowsdetection of the displayed polypeptide at the bacterial cell surface.

A CYTX-DP can display a single DM on either the N-terminus or theC-terminus of the DP. Alternatively, a CYTX-DP can display two DMssimultaneously one on each of the N- and C-termini of the DP. In oneembodiment, a CYTX-DP can display at least two DMs simultaneously, atleast one on either or both of the N- and C-termini of the DP. In someembodiments, a DM is capable of interacting physically with arbitrarycompositions of matter (biological or non-biological), and exhibits abiological activity (e.g., affinity, specificity, catalysis, assemblyetc.) substantially similar to the corresponding free polypeptide insolution. In other words, the displayed DM interacts with or binds agiven target molecule in a manner that is substantially similar to thatwhen the polypeptide is in its native environment and not attached tothe CYTX-CP or a variant thereof.

Biterminal display has numerous advantages, including the ability toquantify the amount of the CYTX-DP displayed on the cell surface and toscreen libraries on both termini simultaneously. For this purpose, aCYTX-DP can include one or more DMs and one or more tags (T) in order toallow detection of surface display. The quantification of the displaylevel during library screening by labeling of a DP allows forpolypeptides with a high affinity but low display level to bedifferentiated from polypeptides with a high display level but moderateaffinity. Moreover, biterminal display allows for the possibility ofcreating peptide libraries on each terminus where both peptides can bindto separate regions of the same protein target, causing increasedbinding affinity and specificity through avidity.

Additionally, linkers may be inserted between a DM of interest andeither the N- or C-terminus of the CYTX-DP to which it is connected inorder to avoid steric hindrance between simultaneously displayed DMsand/or their binding partners. For example, a long flexible linkercomprising multiple repeats of the sequence GGGS (SEQ ID NO: 33) (e.g.,(GGGS)₄ (SEQ ID NO: 34), (GGGS)₅ (SEQ ID NO: 35), or (GGGS)₆ (SEQ ID NO:36)) can be used to increase the accessibility of proteins to ligandsand to avoid steric hindrance when using biterminal display.

Polynucleotides encoding CYTX-DPs of the present invention can beproduced in any number of ways, all of which are well known in the art.

In one embodiment, the polynucleotides are generated using recombinanttechniques, well known in the art. One of skill in the art could readilydetermining nucleotide sequences that encode the desired CYTX-DPs usingstandard methodology and the teachings herein.

Oligonucleotide probes can be devised based on the known sequences ofOmpX proteins and used to probe genomic or cDNA libraries. The sequencescan then be further isolated using standard techniques and, e.g.,restriction enzymes employed to truncate the gene at desired portions ofthe full-length sequence. Similarly, sequences of interest can beisolated directly from cells and tissues containing the same, usingknown techniques, such as phenol extraction and the sequence furthermanipulated to produce the desired CYTX-DPs. See, e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA.

The sequences encoding the CYTX-DPs can also be produced synthetically,for example, based on the known sequences. The nucleotide sequence canbe designed with the appropriate codons for the particular amino acidsequence desired. The complete sequence is generally assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) 1 Biol. Chem.259:6311; Stemmer et al. (1995) Gene 1 164:49-53.

Recombinant techniques are readily used to clone sequences encodingCYTX-DPs useful in the claimed invention that can then be mutagenized invitro by the replacement of the appropriate base pair(s) to result inthe codon for the desired amino acid. Such a change can include aslittle as one base pair, effecting a change in single amino acid, or canencompass several base pair changes. Alternatively, the mutations can beeffected using a mismatched primer that hybridizes to the parentnucleotide sequence (generally cDNA corresponding to the RNA sequence),at a temperature below the melting temperature of the mismatched duplex.The primer can be made specific by keeping primer length and basecomposition within relatively narrow limits and by keeping the mutantbase centrally located. See, e.g., Innis et al, (1990) PCR Applications:Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol.(1983) 100:468. Primer extension is effected using DNA polymerase, theproduct cloned and clones containing the mutated DNA, derived bysegregation of the primer extended strand, selected. Selection can beaccomplished using the mutant primer as a hybridization probe. Thetechnique is also applicable for generating multiple point mutations.See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982)79:6409.

Once coding sequences have been isolated and/or synthesized, they can becloned into any suitable vector or replicon for expression in bacteriaor other cell types as disclosed herein. (See Examples). The inventionalso includes expression constructs for expressing a given DM as anN-terminal fusion protein, a C-terminal fusion protein, or biterminalfusion protein, i.e., linked or fused directly to the CYTX-CP present onthe external surface of a bacterial cell. Display and expression of a DMas an N-terminal or C-terminal or biterminal fusion with a CYTX-DP isaccomplished by topological permutation of an OmpX protein as describedin U.S. patent application Ser. No. 10/920,244, now issued as U.S. Pat.No. 7,256,038, which is herein incorporated by reference. Sequencerearrangement of an OmpX protein can be accomplished using overlapextension PCR methods known in the art in order to create either anN-terminal or C-terminal fusion construct, or alternatively, abiterminal fusion construct. See Ho, et al. (1989) Gene 77(1):51-59,which is herein incorporated by reference. As will be apparent from theteachings herein, a wide variety of vectors encoding CYTX-DPs coupled toone or more DMs can be generated by creating expression constructs thatoperably link, in various combinations, polynucleotides encodingCYTX-DPs and DMs.

Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning include pBAD33, pB30D,pBR322, pACYC177, pKT230, pGV1106, pLAFR1, pME290, pHV14, pBD9, p1161,and pUC6. See, generally, DNA Cloning: Vols. I & II, supra; Sambrook etal., supra; B. Perbal, supra.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired CYTX-DP and DM(s) is transcribed into RNAin the host cell transformed by a vector containing this expressionconstruction. The coding sequence may contain a naturally occurring OmpXsignal peptide sequence or a heterologous signal sequence (e.g., fromanother outer membrane protein such as OmpA, OmpT, OmpC, OmpF, OmpN,LamB, FepA, FecA, or the like) to promote expression of the CYTX-DP atthe surface of a bacterial host cell.

Other regulatory sequences may also be desirable that allow forregulation of expression of the protein sequences relative to the growthof the host cell. Such regulatory sequences are known to those of skillin the art, and examples include those which cause the expression of agene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound (e.g., aregulatable promoter for controlled transcription).

In some embodiments, a vector comprising the regulatable promoter araBADis used to control transcription. Expression and display of thepolypeptide is then accomplished by induction of protein expression bycontacting with arabinose, in some embodiments, for about 10 to about 60minutes, and in some embodiments, for about 10 to about 20 minutes at25° C. Controlling expression and display minimizes potential avidityeffects that can result from excessive surface concentration of thedisplayed peptide.

Expression vectors of the present invention may also utilize a low copyorigin of replication (e.g., p15A) in order to minimize the metabolicburden on the bacterial host cell such that the clonal representation ofthe polypeptide library is not affected by growth competition duringlibrary propagation. Additionally, expression vectors of the presentinvention may include a selectable marker such as an antibacterialresistance gene to a bacteriocidal antibiotic (e.g., chloramphenicolacetyltransferase, beta lactamase, or the like).

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector thatalready contains the control sequences and an appropriate restrictionsite.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. Mutants oranalogs may be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See, e.g., Sambrook et al. supra.

The expression vector is then used to transform an appropriate bacterialhost cell. A number of bacterial hosts are known in the art, includingbut not limited to, Escherichia coli, Shigella sonnei, Shigelladysenteriae, Shigella flexneri, Salmonella typhii, Salmonellatyphimurium, Salmonella enterica, Enterobacter aerogenes, Serratiamarcescens, Yersinia pestis, Bacillus cereus, Bacillus subtilis, orKlebsiella pneumoniae, which will find use with the present expressionconstructs.

In some embodiments, a bacterial strain is chosen that is deficient inproteolytic machinery in order to prevent protein degradation SeeMeerman, H. J., Nature Biotechnol. 12(11):1107-1110, which is hereinincorporated by reference. In some embodiments, a bacterial strain thatmakes truncated or otherwise modified lipopolysaccharides on its surfacemay be used to minimize steric effects upon binding to largebiomolecules including proteins, viruses, cells, and the like. In someembodiments, the bacterial host has a genotype that aids the expressionvector in regulating more tightly the production of the polypeptide tobe displayed. The bacterial host may be modified using methods known inthe art, including random mutagenesis, DNA shuffling, genome shuffling,gene addition libraries, and the like. As exemplified herein,Escherichia coli strain, MC1061 is a suitable bacterial host for displayof DMs using CYTX-DPs of the invention. The MC1061 strain exhibits (1)high transformation efficiency of greater than about 5×10⁹ per microgramof DNA, (2) a short doubling time, i.e., 40 minutes or less, duringexponential growth phase, (3) high level display of the givenpolypeptide, and (4) effective maintenance of the expression ON and OFFstates. Additional bacterial hosts include, but are not limited to, E.coli strains DH-10β, E. coli C41(DE3), E. coli C43(DE3), and E. coli TG1cells.

In some embodiments, the expression vectors and libraries of the presentinvention incorporate (1) the use of a regulatable expression vectorthat allows on-off control of the production of the CYTX-CP or variantthereof, (2) efficient restriction sites immediately adjacent to arandomized site for insertion of cloned DNA encoding a random DM fusedto the N-terminus, C-terminus or both termini of the CYTX-DP tofacilitate library construction, (3) time and temperature-controlledinduction periods to obtain optimal display levels that result in higherquality results, (4) the use of a bacterial strain having a high plasmidtransformation efficiency for transformation, (5) the use of optimizedlibrary construction protocols to construct large libraries, (6) the useof multiple-plasmid transformation to yield a larger number of uniqueDMs for a given number of host cells, (7) the use of cell concentrationto enable complete processing of larger numbers of sequences (e.g.,10¹¹, 10¹², 10¹³, or a larger number depending on sorting capability),or (8) any combination thereof.

In some embodiments of the present invention, a DNA library isconstructed containing greater than about 10⁸ sequences, and in someembodiments, more than about 10¹⁰ unique sequence members, using methodsknown in the art. This library size is used since library size has beenshown to correlate with the quality (affinity and specificity) of theselected sequences. See Griffiths, A. D. and D. S. Tawfik (2000) Curr.Opin. Biotechnol. 11(4):338-53, which is herein incorporated byreference.

In some embodiments, a polypeptide library may be prepared byintroduction and expression of nucleic acid sequences that encodepolypeptides having about 1 to about 1000, in some embodiments, about 2to about 30 amino acids in length. In some embodiments, the polypeptideis greater than 25 amino acids, greater than 50 amino acids, greaterthan 75 amino acids, greater than 100 amino acids, greater than 125amino acids, greater than 150 amino acids, greater than 175 amino acids,greater than 200 amino acids, greater than 225 amino acids, greater than250 amino acids, greater than 275 amino acids, greater than 300 aminoacids long, greater than 350 amino acids long, greater than 400 aminoacids long, or greater than 450 amino acids long. In some embodiments,the polypeptide is no more than 8 amino acids, of no more than 10 aminoacids, of no more than 15 amino acids, of no more than 20 amino acids,of no more than 25 amino acids, of no more than 30 amino acids, of nomore than 35 amino acids, or of no more than 40 amino acids. In certainembodiments, high DNA concentrations of more than about 0.1 ug per ulare used during transformation such that the transformed host cellcontains one or more independent plasmid molecules. Transformation withmultiple plasmids yields a larger number of unique peptides in the samevolume of liquid, providing better overall results than whentransformation is performed with only one molecule per cell. In someembodiments, a mixture of a plurality of different expression vectorsand/or plasmids may be employed, for example, to allow cooperativebinding of two different displayed peptides on the same surface, or topresent a protein having multiple subunits, and the like.

A desired number of DMs, e.g., polypeptides, may be displayed fordifferent purposes. As exemplified herein, the method of the presentinvention utilizes an induction period of about 10 minutes to about 16hours to control total expression levels of the display polypeptide andthe mode of the subsequent screen or selection such that the level ofexpression has no measurable effect upon the cell growth rate. In someembodiments, shorter time periods may be used to reduce avidity effectsin order to allow selection of high affinity monovalent interactions. Asprovided herein, the ability to control display speeds the process andyields higher quality results, e.g., sequences that bind to a targetwith higher affinity.

In some embodiments, a cell concentration by a factor of about 10 may beused to enable complete processing of the entire pool of diversity in avolume of about 10 to about 100 ml. The library may be expanded bypropagation by a factor of more than about 100-fold under conditionsthat prevent synthesis of the library elements, for example, withglucose to repress araBAD or lac promoters, and aliquots of the librarymay be prepared to represent a number of clones that is more than aboutthree fold greater than the total number of library members.

For library selection, a subset of the total library, either randomlydivided, or chosen for specific properties could be used as a startingpoint for screening. Either MACS and/or FACS and/or other suitableselection methods known in the art may be used. Alternatively, methodsknown in the art that enable physical retention of desired clones anddilution or removal of undesired clones may be used. For example, thelibrary may be grown in a chemostat providing continuous growth,diluting out only those cells that do not bind to a capture agentretained in the vessel. Alternatively, hosts may be cultured with mediumhaving ingredients that promote growth of desired clones.

Cell sorting instrumentation is applied as a quantitative libraryscreening tool to isolate the highest affinity clones from amagnetically enriched population. Several different approaches can beapplied for quantitative screening. In some embodiments, screening isbased on the basis of either equilibrium binding affinity (EquilibriumScreen). In some embodiments, screening is based on dissociation rateconstants (Kinetic Screen). In some embodiments, screening is based oncompetitive advantage (Competition Screening) to select for clones thatexhibit superior ligand interaction at increasing concentrations. SeeDaugherty, P. S., et al. (2000) J. Immunol. Methods 243(1-2):211-227;and Boder, E. T. and K. D. Wittrup (1998) Biotechnology Progress14(1):55-62, which are herein incorporated by reference in theirentireties. For equilibrium screening, cell populations are labeled withlimiting concentrations of the target proteins, and all cells exhibitingfluorescence intensities above background autofluorescence arecollected.

Instead of using random synthetic peptides to provide genetic diversity,fragment genomic DNA of varying lengths, cDNA of varying lengths,shuffled DNAs, and consensus generated sequences may be employed inaccordance with the present invention.

Non-natural amino acids having functionality not represented amongnatural amino acids, e.g., metal binding, photoactivity, chemicalfunctionality, and the like, may be displayed on the surface using asuitable bacterial host. In this case, the library or an equivalentlibrary may be transformed into strains engineered to producenon-natural amino acids. See Kiick, K. L. et al. (2001) FEBS Lett.502(1-2):25-30; Kiick, K. L., et al. (2002) PNAS USA 99(1):19-24;Kirshenbaum, K., et al. (2002) Chembiochem. 3(2-3):235-237; and Sharma,N., et al. (2000) FEBS Lett. 467(1):37-40, which are herein incorporatedby reference. Peptides incorporating non-natural amino acids areisolated by selection or screening for functions that require inclusionof the non-natural monomers into the displayed polypeptide.

Displayed polypeptides may be made to include post-translationmodifications, including glycosylation, phosphorylation, hydroxylation,amidation, and the like, by introduction of a gene or set of genesperforming the desired modifications into the strain used for screeningand selection, e.g., MC1061 or comparable host strain. Genes performingsuch post-translational modifications may be isolated from cDNA orgenomic libraries by cotransformation with the library and screening forthe desired function using FACS or another suitable method. For example,post-translational glycosylation activities (enzymes) can be foundco-transforming.

The polypeptides displayed by CYTX-DP or a variant thereof possess alength that preserves the folding and export of the carrier proteinwhile presenting significant sequence and structural diversity. In someembodiments, the CYTX-CP or variant thereof used as a carrier proteinmay be modified by rational redesign or directed evolution by themethods described herein to increase levels of display or enhancepolypeptide presentation. For example, the linker between the native N-and C-termini of OmpX may be optimized by random point or cassettemutagenesis and screened for enhanced presentation. In addition,mutations may be incorporated into the CYTX-CP scaffold that increasethe display efficiency of a DM (e.g., substitutions at positions 165 and166).

Terminal fusion display allows for high mobility of the surfacedisplayed molecule, increased accessibility to target molecules, andsimple proteolytic cleavage of the displayed peptide for production ofsoluble peptides. Terminal fusion display also enables theidentification of novel substrates and ligands, e.g., for proteases,peptidases, kinases, receptors, and antibodies. The expression vectorsaccording to the present invention provide a direct way for enhancingthe conformational diversity and surface mobility of surface anchoredpeptides and polypeptides. Through the increased mobility resulting fromterminal fusion (as opposed to insertional fusions), the apparentaffinity of a polypeptide binding to its corresponding target moleculeor material more closely resembles that of the peptide in solution. TheN-terminal or C-terminal or biterminal display vectors allow theretention of an energetically stable outer membrane protein structure,compatible with folding, transport, and assembly for efficient displayof a given DM on the bacterial cell surface.

In some embodiments, a cDNA library may be cloned into the displayposition of the N-terminal or C-terminal or biterminal fusion expressionvector, with a terminal affinity tag, such as a T7 tag epitope, or alabel, or the like, appended to a terminus of the cDNA clone allowingfor measurement of the total display level on the cell surface. As usedherein, the term “affinity tag” refers to a biomolecule, such as apolypeptide segment, that can be attached to a second biomolecule toprovide for purification or detection of the second biomolecule orprovide sites for attachment of the second biomolecule to a substrate.Examples of affinity tags include a polyhistidine tract, protein A(Nilsson et al. (1985) EMBO J. 4:1075; Nilsson et al. (1991) MethodsEnzymol. 198:3, glutathione S transferase (Smith and Johnson (1988) Gene67:31), Glu-Glu affinity tag (Grussenmeyer et al., (1985) PNAS USA82:7952), substance P, FLAG peptide (Hopp et al. (1988) Biotechnology6:1204), streptavidin binding peptide, or other antigenic epitope orbinding domain, and the like, (Ford et al. (1991) Protein Expression andPurification 2:950), all of which are herein incorporated by reference.As used herein, a “label” is a molecule or atom that can be conjugatedto a biomolecule to render the biomolecule or form of the biomolecule,such as a conjugate, detectable or measurable. Examples of labelsinclude chelators, photoactive agents, radioisotopes, fluorescentagents, paramagnetic ions, and the like.

The presence of surface localized proteins may be monitored using anantibody or reagent specific for the tag or label according to methodsknown in the art. Cells binding to a target protein may be then selectedusing MACS and/or FACS and/or other suitable selection technique(s).

The library pool may be incubated with a fluorescent label of a firstwavelength (such as a label emitting green color) and then a secondfluorescent label of a second wavelength (such as a label emitting redcolor) to identify the presence of a full length cDNA of interest.Clones that exhibit both wavelengths are then isolated from the librarydirectly using cell sorting methods known in the art. In someembodiments, the polypeptides of an N-terminal, C-terminal, orbiterminal fusion expression vector may be isolated or purified from theouter surface of the host. In other words, a polypeptide may beexpressed using an N-terminal, C-terminal, or biterminal fusionexpression vector and then produced in a soluble form (free in solution)by introducing a suppressible codon downstream of the given polypeptide.Alternatively, a protease susceptible linker may be used in place of the“suppressible” codon. The polypeptides are displayed on the surface athigh density by induction, such as with arabinose for a period of about2 hours. The cells are washed once or twice in a compatible buffer, suchas PBS, to remove undesired proteins and other debris, the cells areconcentrated, and a protease is added to the cell suspension. Theproteolytically cleaved polypeptide is then harvested by removal of thebacteria by low-speed centrifugation, and transfer of the supernatantinto a fresh tube.

The present invention provides compositions and methods for screening alibrary of cells presenting DM, e.g., candidate peptides, in peptidedisplay scaffolds to identify a peptide that interacts with an enzyme,where the scaffolds are more resistant to protease degradation. Thesubstrate-resistant cellular libraries of peptide sequences disclosedherein provide a qualitative and/or quantitative approach to identify apeptide ligand for an enzyme as well as determining the specificity ofthe peptide that interacts with an enzyme (e.g., a substrate for theenzyme or an inhibitor of the activity of the enzyme).

In contrast to phagemid or phage libraries displaying DM, e.g.,candidate peptides, the peptide display scaffolds disclosed hereinprovide display of up to about 10³-10⁴ copies of the candidate peptideon the surface of a single cell, thereby enabling identification of apeptide ligand for an enzyme as well as providing for quantitative andqualitative measurement of the interaction between the candidate peptidedisplayed in the peptide display scaffold and the enzyme.

The methods are based on the use of single-cell fluorescence as anindicator of substrate conversion enabling library screening. Likewise,whole-cell fluorescence measurements enable calculation of substratecleavage kinetics for isolated clones, eliminating the need to preparesoluble substrates using synthetic or recombinant methods. Finally, thecell libraries disclosed herein can be manipulated with relative easeand amplified indefinitely by growth without introducing measurablelibrary bias. As such, this approach enables generation of candidatepeptide libraries of arbitrary amino acid compositions and lengths thatare self-renewing. Given the simplicity of library manipulation andscreening, CYTX-DPs provide a scalable solution to rapidly identifycandidate peptides as well as characterize enzymes, such as proteases.

In general, the peptide display scaffolds include a transmembranecarrier protein having N-terminal and C-terminal DM-presenting domainsthat are accessible at a surface of the cell outer membrane, i.e. aredisplayed at the extracellular surface of the cell outer membrane. Thepeptide display scaffolds include at least one N-terminal and C-terminaldomain and at least one N-terminal and C-terminal detectable domains.When expressed in a cell, the peptide display scaffolds display thecandidate peptides as terminal fusion proteins thereby providing a moreaccurate measurement of the interaction capability between the candidatepeptide and the tested enzyme. In other words, the measurement of theinteraction between terminally displayed candidate peptides and enzymesprovided by the present peptide display scaffolds more closelyapproximates values obtained from measurements of the same interactionin solution with soluble peptides. The peptide display scaffolds aredescribed in greater detail below.

The peptide display scaffolds allow the display of DM, e.g., candidatepeptides, at either the N-terminal domain or the C-terminal domain aswell as simultaneous display of a different peptide at each of theN-terminal domain and the C-terminal domain.

In some embodiments, the peptide display scaffolds are generallydescribed by Formula (I) as follows:[T-DM]-CYTX-CP  (I)wherein CYTX-CP is a transmembrane protein that includes at least theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56 or SEQ ID NO: 57, DMis a candidate peptide or other displayed moiety; and T is a detectablemoiety (e.g., affinity tag), wherein when the peptide display scaffoldis expressed in a cell, T-DM is accessible at a surface of the cellouter membrane (e.g., T-DM is on the extracellular surface of the cell).It is to be understood that the T-DM may be at either the N-terminus orthe C-terminus.

In certain embodiments, DM is [S-Ci-] or [Ci-S] and the peptide displayscaffold is generally described by Formula (II) or Formula (III) asfollows:[T-S-C_(i)]-CYTX-CP  (II)[T-C_(i)-S]-CYTX-CP  (III)wherein S is a known substrate for the enzyme, Ci is a candidateinhibitor for the enzyme, CYTX-CP is a transmembrane protein thatincludes at least the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56or SEQ ID NO: 57, and T is a detectable moiety (e.g., affinity tag),wherein when the peptide display scaffold is expressed in a cell,T-S-Cland D-Ci-S is accessible at a surface of the cell outer membrane(e.g., T1-S-Clare T1-Ci-S are on the extracellular surface of the cell).It is to be understood that the T-S-Ci (or T-Ci-S) may be at either theN-terminus or the C-terminus.

In other embodiments, DM is [A-Cs] or [Cs-A] and the peptide displayscaffold is generally described by Formula (IV) or Formula (V) asfollows:[T-A-C_(s)]-CYTX-CP  (IV)[T-C_(s)-A]-CYTX-CP  (V)wherein A is an allosteric regulator for the enzyme, Cs is a candidatesubstrate for the enzyme, CYTX-CP is a transmembrane protein thatincludes at least the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56or SEQ ID NO: 57, and T is a detectable moiety (e.g., affinity tag),wherein when the peptide display scaffold is expressed in a cell,T1-A-Cs and T1-Cs-A are accessible at a surface of the cell outermembrane (e.g., T1-A-Cs and T1-C_(s)-A are on the extracellular surfaceof the cell). It is to be understood that the T1-A-Cs (or T1-Cs-A) maybe at either the N-terminus or the C-terminus.

In some embodiments, the peptide display scaffolds are generallydescribed by Formula (VI) as follows:[T₁-DM]-CYTX-CP-[T2]  (VI)wherein CYTX-CP is a transmembrane protein that includes at least theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56 or SEQ ID NO: 57, DMis a candidate peptide; and T₁ and T2 are first and second detectablemoieties (e.g., affinity tags), wherein T1 and T2 are different andwherein when the peptide display scaffold is expressed in a cell, T1-DMand T2 are accessible at a surface of the cell outer membrane (e.g.,T1-DM and T2 are on the extracellular surface of the cell) and whereinwhen T1 is present (e.g., provides a detectable signal by binding of anaffinity ligand labeled fluorescent moiety) T2 does not provide adetectable signal; in some embodiments, both T1 and T2 providedetectable signals (FIG. 3, panels A and B). It is to be understood thatthe T1-DM may be at either the N-terminus or the C-terminus and T2 maybe at either the N-terminus or the C-terminus. For example, when theT1-DM is at the N-terminus the T2 is at the C-terminus (FIG. 3, panel A)and when the T1-DM is at the C-terminus the T2 is at the N-terminus(FIG. 3, panel B).

In certain embodiments, DM is [S-C_(i)] or [Ci-S} and the peptidedisplay scaffold is generally described by Formula (VII) or Formula(VIII) as follows:[T1-S-C_(i)]-CYTX-CP-[T2]  (VII)[T1C_(i)-S]-CYTX-CP-[T2]  (VIII)wherein S is a substrate for the enzyme, Ci is a candidate inhibitor forthe enzyme, CYTX-CP is a transmembrane protein that includes at leastthe amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56 or SEQ ID NO: 57,and T1 and T2 are first and second detectable moieties (e.g., affinitytags), wherein T1 and T2 are different and wherein when the peptidedisplay scaffold is expressed in a cell, T1-Ci-S, and T2 are accessibleat a surface of the cell outer membrane (e.g., T1-S-C1, T1-Ci-S, and T2are on the extracellular surface of the cell) and wherein when T1 ispresent (e.g., provides a detectable signal by binding of an affinityligand labeled fluorescent moiety) T2 does not provide a detectablesignal; in some embodiments, both T1 and T2 provide detectable signals.(FIG. 3, panels C, D, E, and F). It is to be understood that the T1-S-Ci(or T1-Ci-S) may be at either the N-terminus or the C-terminus and T2may be at either the N-terminus or the C-terminus. For example, when theT1-S-C1 (or T1-Ci-S) is at the N-terminus the T2 is at the C-terminus(FIG. 3, panels C and D) and when the T1-S-Ci (or T1-Ci-S) is at theC-terminus the T2 is at the N-terminus (FIG. 3, panels E and F).

In other embodiments, DM is [A-Cs] or [Cs-A] and the peptide displayscaffold is generally described by Formula (IX) or Formula (X) asfollows:[T1-A-C_(s)]-CYTX-CP-[T2]  (IX)[T1-C_(s)-A]-CYTX-CP-[T2]  (X)wherein A is an allosteric regulator for the enzyme, C_(s) is acandidate substrate for the enzyme, CYTX-CP is a transmembrane proteinthat includes at least the amino acid sequence of SEQ ID NO: 1, SEQ IDNO: 56 or SEQ ID NO: 57, and T1 and T2 are first and second detectablemoieties (e.g., affinity tags), wherein T1 and T2 are different andwherein when the peptide display scaffold is expressed in a cell,T1-A-Cs, T1-Cs-A, and T2 are accessible at a surface of the cell outermembrane (e.g., T1-A-Cs, T1-Cs-A, and T2 are on the extracellularsurface of the cell) and wherein when T1 is present T2 does not providea detectable signal; in some embodiments, both T1 and T2 providedetectable signals (FIG. 4, panels A, B, C, and D). It is to beunderstood that the T1-A-Cs (or T1-Cs-A) may be at either the N-terminusor the C-terminus and T2 may be at either the N-terminus or theC-terminus. For example, when the T1-A-Cs (or T1-Cs-A) is at theN-terminus the T2 is at the C-terminus (FIG. 4, panels A and B) and whenthe T1-A-Cs (or T1-C_(s)-A) is at the C-terminus the T2 is at theN-terminus (FIG. 4, panels C and D).

In other embodiments, the peptide display scaffolds are generallydescribed by Formula (XI) as follows:[T1-C₁]-CYTX-CP-[C₂-T2]  (XI)wherein CYTX-CP is a transmembrane protein that includes at least theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56 or SEQ ID NO: 57, C1and C2 are first and second candidate peptides, wherein C1 and C2 arenot the same; T1 and T2 are first and second detectable moieties (e.g.,affinity tags), wherein T1 and T2 are not the same; and wherein when thepeptide display scaffold is expressed in a cell, T1-C1 and C2-T2 areaccessible at a surface of the cell outer membrane (e.g., T1-C1 andC2-T2 are on the extracellular surface of the cell) (FIG. 5, panels Aand B). It is to be understood that the T1-C1 may be at either theN-terminus or the C-terminus and C2-T2 may be at either the N-terminusor the C-terminus. For example, when the T1-C1 is at the N-terminus theC2-T2 is at the C-terminus (FIG. 5, panel A) and when the T1-C1 is atthe C-terminus the C2-T2 is at the N-terminus (FIG. 5, panel B). Incertain embodiments, C1 is an allosteric regulator and C2 is a candidatesubstrate (FIG. 5, panels C and D). In other embodiments, C1 is a knownsubstrate and C2 is a candidate inhibitor (FIG. 5, panels E and F).

Exemplary transmembrane proteins (CYTX-CP) and methods for modifying thesame for use with the peptide display scaffolds are described in greaterdetail in the Examples provided herein. It should be noted that anytransmembrane protein localized on the outer surface of a biologicalentity, presenting one or more loop sequences accessible on the cellsurface and the like may be modified in order to generate and present aC-terminus, an N-terminus, or both at the outer surface of a biologicalentity and fused with a DM is suitable for use with the peptide displayscaffolds.

In certain embodiments, the peptide display scaffolds further include aflexible linker between the transmembrane protein (CYTX-CP) and one orboth of the N-terminal and C-terminal domains, such as T1-DM, T1-Ci-S,T1-A-Cs, T1-C_(s)-A, T2, T1-C1 and C2-T2. For example, in someembodiments, the peptide display scaffold further includes a linkerbetween DM and CYTX-CP or at least one linker between C₁ and CYTX-CP orC2 and CYTX-CP.

A linker suitable for use with the peptide display scaffold will be onethat provides flexibility to the N-terminal or C-terminal domains whenpresent and does not interfere with the presentation of the candidatepeptide on the surface of the cell. The flexible linker will be variablelength, such as from about 3 amino acids to about 25 amino acids,including about 4 amino acids to about 23 amino acids, about 5 aminoacids to about 20 amino acids, about 6 amino acids to about 18 aminoacids, about 7 amino acids to about 16 amino acids, about 8 amino acidsto about 14 amino acids, and about 9 amino acids to about 12 aminoacids.

Exemplary flexible linkers include glycine polymers (G)., glycine-serinepolymers (including, for example, (GS)_(n), (GSGGS) (SEQ ID NO: 37) and(GGGS) (SEQ ID NO: 34), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers such as the tether for the shaker potassium channel, and a largevariety of other flexible linkers, as will be appreciated by those inthe art. Glycine and glycine-serine polymers are of interest since bothof these amino acids are relatively unstructured, and therefore may beable to serve as a neutral tether between components. Glycine polymersare of particular interests glycine accesses significantly more phi-psispace than even alanine, and is much less restricted tan residues withlonger side chains (see Scheraga, Rev. Computational Chem. 11173-142(1992)). Exemplary flexible linkers include, but are not limitedGly-Gly-Ser-Gly-Gly (SEQ ID NO: 38), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:39), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 40), Gly-Gly-Gly-Ser-Gly (SEQ IDNO: 41), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 42), and the like.

As described above, the candidate peptides (C) are generally situated ona cell surface accessible region of a peptide display scaffold, suchthat the candidate peptides can interact with extracellular or cellsurface-associated elements. The candidate peptides can be screened toidentify a peptide ligand for the tested enzyme. As used herein,“ligand” or “peptide ligand” refer to a molecule(s) that binds (e.g., bycovalent or non-covalent interaction) to a binding partner molecule(s),e.g., a substrate, inhibitor, or allosteric regulator binding to anenzyme. The binding of the ligand to the binding partner may be at theactive site, e.g., binding of a substrate or inhibitor with an enzyme,or at another secondary site e.g., binding of an allosteric regulator ornon-competitive inhibitor with an enzyme. As such, exemplary candidatepeptides include candidate enzyme substrates, candidate enzymeinhibitors, and the like.

Candidate peptides can range from about 2 amino acids in length to about500 amino acids, including polypeptides ranging from about 2 to about450, from about 2 to about 400, from about 2 to about 350, from about 2to about 300, such as from about 2 to about 250 amino acids in length,from about 2 to about 200 amino acids in length, from about 2 to about150 amino acids in length, from about 2 to about 100 amino acids inlength, from about 2 to about 50 amino acids in length, from about 2 toabout 40 amino acids in length, from about 2 to about 30 amino acids inlength, from about 2 to about 25 amino acids in length, from about 2 toabout 20 amino acids in length, from about 2 to about 15 amino acids inlength, from about 2 to about 10 amino acids in length, being suitable.For example, in some embodiments, the candidate polypeptide is a singlechain Fv (scFv) or other antibody fragment. In some embodiments whereinthe candidate polypeptide is an antibody fragment, the antibody fragmentis at least 50, at least 100, at least 150, at least 200, at least 250,at least 300, at least 350, at least 400, at least 450, or at least 500amino acids in length. In some embodiments the antibody fragment is ascFv. In some embodiments the antibody fragment is a scFab or splitchain Fab at different fusion locations. In some embodiments, thecandidate polypeptide is a substrate. In some embodiments wherein thecandidate polypeptide is a substrate, the substrate is no more than 15,no more than 10, or no more than 8 amino acids in length. In someembodiments, the candidate polypeptide is a masking moiety. In someembodiments wherein the candidate polypeptide is a masking moiety, themasking moiety is no more than 40 amino acids in length,

In general, the candidate peptide are randomized, either fullyrandomized or are biased in their randomization, e.g., innucleotide/residue frequency generally or per position. By “randomized”is meant that each candidate peptide consists of essentially randomamino acids. As is more fully described below, the candidate peptides,or candidate nucleic acids encoding the same, are chemicallysynthesized, and thus may incorporate any amino acid or nucleotide atany position. The synthetic process can be designed to generaterandomized peptides, to allow the formation of all or most of thepossible combinations over the length of the peptide, thus forming alibrary of randomized candidate peptides.

As such, in some embodiments, the library of candidate peptides is fullyrandomized, with no sequence preferences or constants at any position.In other embodiments, the library of candidate peptides is biased. Thatis, some positions within the sequence are either held constant, or areselected from a limited number of possibilities. For example, in oneembodiment, the nucleotides or amino acid residues are randomized withina defined class, for example, of hydrophobic amino acids, hydrophilicresidues, sterically biased (either small or large) residues, towardsthe creation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,etc., or to purines, etc.

The first and second detectable moieties/tags (T1 and T2) can be anydetectable label/tag that provides a detectable signal that can beassessed qualitatively (positive/negative) and quantitatively(comparative degree of fluorescence). As noted in greater detail above,the first and second detectable moieties (T1 and T2) of a peptidedisplay scaffold are different. As used herein, the terms “label” and“detectable label” refer to a molecule capable of detection, including,but not limited to, radioactive isotopes, fluorescers, chemiluminescers,chromophores, enzymes, enzyme substrates, enzyme cofactors, enzymeinhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g.,biotin, avidin, streptavidin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof that is capableof exhibiting fluorescence in the detectable range. Exemplary detectablemoieties suitable for use with the peptide display scaffolds includeaffinity tags and fluorescent proteins.

The term “affinity tag” is used herein to denote a peptide segment thatcan be attached to peptide display scaffolds at position T (e.g., T1 orT2) that can be detected using a molecule that binds the affinity tagand provides a detectable signal (e.g., a fluorescent compound orprotein). In principal, any peptide or protein for which an antibody orother specific binding agent is available can be used as an affinitytag. Exemplary affinity tags suitable for use include, but are notlimited to, a monocytic adaptor protein (MONA) binding peptide, a T7binding peptide, a streptavidin binding peptide, a polyhistidine tract,protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith andJohnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al.,Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide(Hopp et al., Biotechnology 6:1204 (1988)), or other antigenic epitopeor binding domain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

Any fluorescent polypeptide (also referred to herein as a fluorescentlabel) well known in the art is suitable for use as a detectable moietyor with an affinity tag of the peptide display scaffolds describedherein. A suitable fluorescent polypeptide will be one that can beexpressed in a desired host cell, such as a bacterial cell or amammalian cell, and will readily provide a detectable signal that can beassessed qualitatively (positive/negative) and quantitatively(comparative degree of fluorescence). Exemplary fluorescent polypeptidesinclude, but are not limited to, yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), GFP, mRFP, RFP (tdimer2), HCRED, etc., or anymutant (e.g., fluorescent proteins modified to provide for enhancedfluorescence or a shifted emission spectrum), analog, or derivativethereof. Further suitable fluorescent polypeptides, as well as specificexamples of those listed herein, are provided in the art and are wellknown.

Also described herein are nucleic acid compositions encoding the peptidedisplay scaffolds described herein. For example, the nucleic acidmolecules encode the peptide display scaffolds of Formulas VI-XI.Nucleic acid compositions of particular interest comprise a sequence ofDNA having an open reading frame that encodes a peptide display scaffoldand is capable, under appropriate conditions, of being expressed andprovide display of the candidate peptide at the extracellular surface ofthe cell outer membrane.

In certain embodiments, the nucleic acid encoding the peptide displayscaffolds of Formulas VI-X may further include at least one restrictionendonuclease site (e.g., a single endonuclease site or a multiplecloning site (e.g., polylinker)) between T1 and DM (or Cl, S, Cs, A) andat least one restriction endonuclease site (e.g., a single endonucleasesite or a multiple cloning site (e.g., polylinker)) between CYTX-CP andT2. In other embodiments, the nucleic acid encoding the peptide displayscaffold of Formula XI may further include at least one restrictionendonuclease site (e.g., a single endonuclease site or a multiplecloning site (e.g., polylinker)) between T1 and C1 and at least onerestriction endonuclease site (e.g., a single endonuclease site or amultiple cloning site (e.g., polylinker)) between T2 and C2. Alsoencompassed in this term are nucleic acids that are homologous,substantially similar or identical to the nucleic acids disclosedherein.

In certain embodiments, the nucleic acids may be present in anappropriate vector for extrachromosomal maintenance or for integrationinto a host genome, as described in greater detail below.

In some embodiments, the vector includes a nucleic acid encoding apeptide display scaffold generally described by Formula (XII) asfollows:[T1-RE]-CYTX-CP-[T2]  (XII)wherein CYTX-CP is a transmembrane protein that includes at least theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56 or SEQ ID NO: 57, REis a restriction endonuclease site for insertion of a nucleic acidsequence encoding a candidate peptide; and T1 and T2 are first andsecond detectable moieties (e.g., affinity tags), wherein T1 and T2 aredifferent.

In certain embodiments, the vector includes a nucleic acid encoding apeptide display scaffold generally described by Formula (XIII) orFormula (XIV) as follows:[T1-Y-RE]-CYTX-CP-[T2]  (XIII)[T1-RE-Y]-CYTX-CP-[T2]  (XIV)wherein Y is a substrate for the enzyme or an allosteric regulator forthe enzyme that includes at least the amino acid sequence of SEQ ID NO:1, SEQ ID NO: 56 or SEQ ID NO: 57, RE is a restriction endonuclease sitefor insertion of a nucleic acid sequence encoding a candidate inhibitorfor the enzyme, CYTX-CP is a transmembrane protein, and T1 and T2 arefirst and second detectable moieties (e.g., affinity tags), wherein T1and T2 are different.

In other embodiments, the peptide display scaffolds are generallydescribed by Formula (XV) as follows:[T1-RE₁]-CYTX-CP-[RE₂-T2]  (XV)wherein CYTX-CP is a transmembrane protein that includes at least theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 56 or SEQ ID NO: 57, RE1and RE₂ are first and second restriction endonuclease sites, and T1 andT2 are first and second detectable moieties (e.g., affinity tags),wherein T1 and T2 are different and RE1 and RE2 are different.

Any restriction endonuclease site can be used at RE, RE₁, and RE₂ thatprovides for efficient restriction and insertion of a nucleic acidencoding a candidate peptide is suitable for use. Exemplary restrictionendonuclease sites suitable for use include, but are not limited to,EagI, Not1, BamH1, Hind3, EcoR1, Hpl1, Sal1, Sfi1, Cla1, Rsr2, and thelike.

It will be appreciated that in some embodiments it will be desirable touse a single polylinker having at least two more different endonucleasesites at the RE, RE1, and RE2. In such embodiments, the expressionvector includes a polylinker at RE, RE1, and RE2 having at least two ormore different restriction endonuclease sites (e.g., multiple cloningsite). For example, in such embodiments, the vectors encoding thepeptide display scaffold includes a polylinker having two or more sitesto provide for insertion of a nucleic acid sequence encoding a candidatepeptide using a first restriction endonuclease site and allow forexcision of the nucleic acid once a specific clone that has beenidentified as being of particular interest using two flankingrestriction endonuclease sites.

The polynucleotides and constructs thereof can be generatedsynthetically by a number of different protocols known to those of skillin the art. Appropriate polynucleotide constructs are purified usingstandard recombinant DNA techniques as described in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989)Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and under currentregulations described in United States Dept. of HEIS, National Instituteof Health (NIH) Guidelines for Recombinant DNA Research.

Also provided are constructs comprising the nucleic acids describedherein inserted into a vector, where such constructs may be used for anumber of different screening applications as described in greaterdetail below. In some embodiments, a single vector (e.g., a plasmid)will contain nucleic acid coding sequence for a single peptide displayscaffold. In other embodiments, a single vector (e.g., a plasmid) willcontain nucleic acid coding sequence for a two or more peptide displayscaffolds.

Viral and non-viral vectors may be prepared and used, includingplasmids, which provide for replication of biosensor-encoding DNA and/orexpression in a host cell. The choice of vector will depend on the typeof cell in which propagation is desired and the purpose of propagation.Certain vectors are useful for amplifying and making large amounts ofthe desired DNA sequence. Other vectors are suitable for expression incells in culture. Still other vectors are suitable for transformationand expression in cells in a whole animal or person. The choice ofappropriate vector is well within the skill of the art. Many suchvectors are available commercially. To prepare the constructs, thepartial or full-length polynucleotide is inserted into a vectortypically by means of DNA ligase attachment to a cleaved restrictionenzyme site in the vector. Alternatively, the desired nucleotidesequence can be inserted by homologous recombination in vivo. Typicallythis is accomplished by attaching regions of homology to the vector onthe flanks of the desired nucleotide sequence. Regions of homology areadded by ligation of oligonucleotides, or by polymerase chain reactionusing primers comprising both the region of homology and a portion ofthe desired nucleotide sequence, for example.

Also provided are expression cassettes or systems that find use in,among other applications, the synthesis of the peptide displayscaffolds. For expression, the gene product encoded by a polynucleotideof the invention is expressed in any convenient expression system,including, for example, bacterial, yeast, insect, amphibian andmammalian systems. Suitable vectors and host cells are described in U.S.Pat. No. 5,654,173. In the expression vector, a polynucleotide is linkedto a regulatory sequence as appropriate to obtain the desired expressionproperties. These regulatory sequences can include promoters (attachedeither at the 5′ end of the sense strand or at the 3′ end of theantisense strand), enhancers, terminators, operators, repressors, andinducers. The promoters can be regulated or constitutive. In somesituations it may be desirable to use conditionally active promoters,such as tissue-specific or developmental stage-specific promoters. Theseare linked to the desired nucleotide sequence using the techniquesdescribed above for linkage to vectors. Any techniques known in the artcan be used. In other words, the expression vector will provide atranscriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be native to the species from which thenucleic acid is obtained, or may be derived from exogenous sources.

Eukaryotic promoters suitable for use include, but are not limited to,the following: the promoter of the mouse metallothionein I gene sequence(Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter ofHerpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter(Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall genesequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA)81:5951-59SS, 1984), the CMV promoter, the EF-1 promoter,Ecdysone-responsive promoter(s), tetracycline-responsive promoter, andthe like.

Promoters may be, furthermore, either constitutive or regulatable.Inducible elements are DNA sequence elements that act in conjunctionwith promoters and may bind either repressors (e.g., lacO/LAC Iqrepressor system in E. coli) or inducers (e.g., gall/GAL4 inducer systemin yeast). In such cases, transcription is virtually “shut off” untilthe promoter is derepressed or induced, at which point transcription is“turned-on.”

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor, among other things, the screening methods described in greaterdetail below.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. After introduction of the DNA, the cells containingthe construct may be selected by means of a selectable marker, the cellsexpanded and then used for expression.

The above described expression systems may be employed with prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. In some embodiments, a unicellular organism,such as E. coli, B. subtilis, S. cerevisiae, insect cells in combinationwith baculovirus vectors, or cells of a higher organism such asvertebrates, e.g., COS 7 cells, HEK 293, CHO, Xenopus Oocytes, etc., maybe used as the expression host cells. In other situations, it isdesirable to use eukaryotic cells, where the expressed protein willbenefit from native folding and post-translational modifications.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Expression systemsin bacteria include those described in Chang et al., Nature (1978)275:615; Goeddel et al., Nature (1979) 281:544; Goeddel et al., NucleicAcids Res. (1980) 8:4057; EP 0 036,776; U.S. Pat. No. 4,551,433; DeBoeret al., Proc. Natl. Acad. Sci. (USA) (1983) 80:21-25; and Siebenlist etal., Cell (1980) 20:269.

Mammalian expression is accomplished as described in Dijkema et al.,EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad. Sci. (USA) (1982)79:6777, Boshart et al., Cell (1985) 41:521 and U.S. Pat. No. 4,399,216.Other features of mammalian expression are facilitated as described inHam and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal.Biochem. (1980) 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.

As will be appreciated by those in the art, the type of host cellssuitable for use can vary widely. In some embodiments, the cell is abacterial cell, a yeast cell or a mammalian cell. In some embodiments,the biological entity is a bacterial cell. In some embodiments, thebacterial cell is Escherichia coli, Shigella sonnei, Shigelladysenteriae, Shigella flexneri, Salmonella typhii, Salmonellatyphimurium, Salmonella enterica, Enterobacter aerogenes, Serratiamarcescens, Yersinia pestis, Bacillus cereus, Bacillus subtilis, orKlebsiella pneumoniae.

The constructs can be introduced into the host cell by any one of thestandard means practiced by one with skill in the art to produce a cellline of the invention. The nucleic acid constructs can be delivered, forexample, with cationic lipids (Goddard, et al, Gene Therapy,4:1231-1236, 1997; Gorman, et al, Gene Therapy 4:983-992, 1997;Chadwick, et al, Gene Therapy 4:937-942, 1997; Gokhale, et al, GeneTherapy 4:1289-1299, 1997; Gao, and Huang, Gene Therapy 2:710-722, 1995,all of which are incorporated by reference herein), using viral vectors(Monahan, et al, Gene Therapy 4:40-49, 1997; Onodera, et al, Blood91:30-36, 1998, all of which are incorporated by reference herein), byuptake of “naked DNA”, and the like.

Also disclosed herein are cellular libraries of candidate peptidesequences including a plurality of cells each expressing a peptidedisplay scaffold and presenting at least one candidate peptide. By a“plurality of cells” or a “population of host cells” herein is meantroughly from about 10³ cells to 10¹¹ or 10¹³ cells. In some embodiments,such a cellular library comprises from 10³ to 10⁵ cells. In someembodiments, such a cellular library comprises from 10⁴ to 10⁶ cells. Insome embodiments, such a cellular library comprises from 10⁵ to 10⁷cells. In some embodiments, such a cellular library comprises from 10⁶to 10⁸ cells. In some embodiments, such a cellular library comprisesfrom 10⁷ to 10⁹ cells. In some embodiments, such a cellular librarycomprises from 10⁸ to 10¹⁰ cells. In some embodiments, such a cellularlibrary comprises from 10⁹ to 10¹¹ cells. In some embodiments, such acellular library comprises from 10¹⁰ to 10¹² cells. In some embodiments,such a cellular library comprises from 10¹¹ to 10¹³ cells. In someembodiments, such a cellular library is of any size depending on sortingcapability.

This plurality of cells comprises a cellular library, wherein generallyeach cell within the library includes at least one peptide displayscaffold at the outer membrane. In certain embodiments, the library isenriched for cells expressing peptide display scaffolds presentingcandidate peptides. By “enriched” is meant that the cells of the libraryexhibit at least one detectable signal from the peptide displayscaffolds. The enrichment of the cells cane done by, for example,fluorescence activated cell sorting.

In some embodiments, each cell of the cellular library expresses asingle type of peptide display scaffold. For example, each cellexpresses at least one peptide display scaffold on the extracellularsurface of the cell outer membrane, wherein all the peptide displayscaffolds of the cell present the same candidate peptide.

In other embodiments, the cellular library includes cells expressing twoor more different types of peptide display scaffolds, including three ormore and four or more, etc. By “different types of peptide displayscaffolds” is meant that each type of peptide display scaffold displayedon the surface of the cell presents a candidate peptide that isdifferent than the candidate peptide presented by the other type ofpeptide display scaffold displayed on the surface of the cell. Forexample, in embodiments in which a cellular library includes a cellexpressing a first and second peptide display scaffold, the candidatepeptide presented by the first peptide display scaffold is differentfrom the candidate peptide presented by the second peptide displayscaffold. It will be appreciated by one of skill in the art that in suchembodiments, the T1 and T2 of the first peptide display scaffold will bedifferent than the T1 and T2 of the second peptide display scaffold.

In one embodiment, the CYTX-DP is a library of fully randomizedcandidate peptides, with no sequence preferences or constants at anyposition. In another embodiment, the CYTX-DP is a library of biasedcandidate peptides. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in one embodiment, the nucleotides or aminoacid residues are randomized within a defined class, for example, ofhydrophobic amino acids, hydrophilic residues, sterically biased (eithersmall or large) residues, towards the creation of cysteines, forcross-linking, prolines for SH-3 domains, serines, threonines, tyrosinesor histidines for phosphorylation sites, etc., or to purines, etc.

In another embodiment, the bias is towards peptides that interact withknown classes of enzymes, e.g., proteases. A number of molecules orprotein domains are suitable as starting points for the generation ofbiased randomized candidate regulator polypeptides. A large number ofsmall molecule domains are known that confer a common function,structure or affinity In addition, as is appreciated in the art, areasof weak amino acid homology may have strong structural homology. Anumber of these molecules, domains, and/or corresponding consensussequences, are known, including, but are not limited to, SH-2 domains,SH-3 domains, Pleckstrin, death domains, protease cleavage/recognitionsites, enzyme inhibitors, enzyme substrates, Traf, etc. In addition,agonists and antagonists of any number of molecules may be used as thebasis of biased randomization of candidate regulator polypeptides aswell.

Applications

The present invention may be broadly applied to methods to isolate,improve or otherwise alter, peptide and polypeptide sequences thatperform useful or desired functions including binding, catalysis,assembly, transport, and the like. For example, the CYTX-DPs of thepresent invention may be used to isolate peptide moleculartransformation catalysts, develop whole-cell reagents, discover peptidesthat promote self-assembly, discover in vivo targeting peptides for drugand gene delivery, discover and improve peptides binding to materialssurfaces, e.g., semiconductors, mapping proteins such as proteincontacts, and biomolecular networks, identifying enzyme substratesand/or inhibitors, identifying receptor agonists and/or antagonists,isolating inhibitors of bacterial or viral pathogenesis, discoveringpeptides that mediate endocytosis and cellular entry, mapping antibodyand protein epitopes including multiplex mapping, identifying peptidemimics of non-peptide ligands, isolating metal binding peptides, e.g.,for bioremediation, nano-wire synthesis, according to methods known inthe art, and modifying antibodies or other proteins. See Georgiou, G.,et al. (1997) Nat. Biotechnol. 15(1):29-34; Pasqualini, R. and E.Ruoslahti (1996) Nature 380(6572):364-366; Whaley, S. R., et al. (2000)Nature 405(6787):665-668; Fields, S, and R. Sternglanz (1994) Trends inGenetics 10(8):286-292; Kim, W. C., et al. (2000) J. Biomol. Screen.5(6):435-440; Yang, W. P., et al. (1995) J. Mol. Biol. 254(3): 392-403;Poul, M. A., et al. (2000) J. Mol. Biol. 301(5):1149-1161; James, L. C.,et al. (2003) Science 299(5611):1362-1367; Feldhaus, M. J., et al.(2003) Nat. Biotechnol. 21(2):163-170; Kjaergaard, K., et al. (2001)Appl. Environ. Microbiol. 67(12):5467-5473, and Shusta, E. V., et al.(1999) Curr. Opin. Biotechnol. 10(2):117-122, which are hereinincorporated by reference.

As provided herein, the CYTX-DPs of the present invention may be used toelucidate consensus sequences while maintaining diversity in selectedpopulations according to methods known in the art. See Smith, G. P. andA. M. Fernandez (2004) Biotechniques 36(4):610-614, 616, 618; andLowman, H. B. (1997) Ann. Rev. Biophys. Biomol. Struct. 26:401-424,which are herein incorporated by reference.

In some embodiments, the DMs may increase the frequency at which highaffinity binders occur relative to the prior art that enables longerconsensus motifs and secondary structures to be determined. SeeNakamura, G. R., et al. (2002) PNAS USA 99(3):1303-1308, which is hereinincorporated by reference.

The CYTX-DPs of the present invention used in conjunction with FACSprovides fine discrimination of clonal affinity, and quantitativeseparations that take advantage of this sensitivity. See Van Antwerp, J.J. and K. D. Wittrup (2000) Biotechnol. Prog. 16(1):31-37; andDaugherty, P. S., et al. (1998) Protein Eng. 11(9):825-832, which areherein incorporated by reference. Specifically, the fine affinitydiscrimination provided by FACS allowed isolation of the best sequencesbinding to streptavidin, CRP, and anti-T7•tag Mab. Further, the displaysystems herein routinely enabled identification of beneficial cysteineplacements to form putative disulfide constrained loops conferring highbinding affinity without explicit library design, which alleviates theneed to construct and screen twenty or more different libraries, andremoves critical assumptions that have limited the affinities ofisolated ligands in earlier studies. See Giebel, L. B., et al. (1995)Biochemistry 34(47): 15430-15435; Deshayes, K., et al. (2002) Chem.Biol. 9(4):495-505; and Nakamura, G. R., et al. (2002) PNAS USA99(3):1303-1308, which are herein incorporated by reference.

The present invention provides construction of a single library ofsufficient size and quality enables routine isolation of high affinitycyclic peptides. For the construction of intrinsically fluorescentlibraries, a ribosomal binding site (RBS) known in the art may beintroduced downstream of the carrier protein, e.g., CYTX-CP, followed bya suitable fluorescent protein, e.g., alaj GFP. See Bessette, P. H. andP. S. Daugherty (2004) Biotechnology Progress 20 (1), which is hereinincorporated by reference. The resulting bacteria, when expression isinduced by the addition of 0.2% arabinose, are both intrinsicallylabeled and display DMs as N- or C-terminal fusion proteins.Alternatively, the order may be reversed such that the fluorescentprotein is expressed first, followed by the RBS and the permuted OMPsequence.

Sequences with about 10 to about 100 fold higher affinity may beobtained by randomization of non-consensus residues and kinetic FACSselection (using biotin as a competitor). Streptavidin binding peptidesmay be used as genetically encoded biotin mimics to eliminate the needfor chemical labeling of proteins with biotin. Thus, a streptavidinbinding peptide selected and affinity matured using this process couldbe fused, using recombinant methods known in the art, to either the C orN-terminus of at least one given nucleic acid molecule. Expression ofthe nucleic acid molecule would produce a polypeptide having a C- orN-terminal peptide tag capable of binding to the commonly used affinityreagent, streptavidin, which may be eluted from the reagent by thesimple addition of biotin.

The polypeptide display systems of the present invention allow thecreation of renewable whole cell binding reagents in non-specializedlaboratories since this method is technically accessible and librariesare reusable. This approach has already proven useful for selectingcell-specific binding peptides, and for performing diagnostic assaysusing flow cytometry and fluorescence microscopy (unpublished data).Furthermore, the surface displayed polypeptides can be used for parallelor multiplex ligand isolation, and clones can be processed withefficient single-cell deposition units present on many cell sorters. SeeFeldhaus, M. J., et al. (2003) Nat. Biotechnol. 21(2):163-170, which isherein incorporated by reference. Consequently, the CYTX-DPs of thepresent invention may be used in proteomic applications includingproteome-wide ligand screens for protein-detecting array development.See Kodadek, T. (2001) Chem. Biol. 8 (2): 105-115, which is hereinincorporated by reference.

In one embodiment, CYTX-DPs of the invention can be used in displaylibraries for screening polypeptides for biological activity. Apolypeptide display library, as described herein, is provided comprisingCYTX-DPs carrying a plurality of DMs displayed on bacterial cells. Thepolypeptides are contacted with a target molecule of interest andassayed for biological activity in the presence of the target moleculein order to identify displayed DMs that have biological activity. Forthis purpose, any CYTX-DP described herein can be used in thepolypeptide display library for screening polypeptides. The polypeptidedisplay library can include DMs fused to the N- or C- or both termini ofthe CYTX-DPs. The biological activity assayed can be enzymatic activity,substrate activity, ligand-binding activity, agonist activity,antagonist activity, transport activity, or any other biologicalactivity. Any target molecule can be chosen, including but not limitedto, a receptor, a ligand, an antibody, an antigen, an enzyme, atransporter, a substrate, an inhibitor, an activator, a cofactor, adrug, a nucleic acid, a lipid, a carbohydrate, a glycoprotein, a smallorganic molecule, or an inorganic molecule.

As used herein, “interact” or “interaction” with respect to a candidatepeptide and an enzyme is meant the recognition and involvement betweenthe enzyme and peptide to produce an effect either on the peptide or theenzyme. For example, “interaction” includes cleavage of a candidatesubstrate by the enzyme, inhibition of an enzyme by a candidateinhibitor, modulation of enzyme specificity and/or activity by acandidate allosteric regulator, modulation of enzyme specificity and/oractivity with respect to a candidate peptide (e.g., candidate substrateor candidate inhibitor) by a known allosteric regulator, and the like.As such, exemplary candidate peptides include candidate enzymesubstrates, candidate enzyme inhibitors, candidate allosteric regulatorsof enzymes, and the like.

In certain embodiments, the invention includes a method of screening alibrary of polypeptides for the ability to bind to a target molecule,the method comprising: a) providing a polypeptide display librarycomprising CYTX-DPs carrying a plurality of DMs displayed on bacterialcells, b) contacting the plurality of DMs with the target molecule, andc) identifying at least one displayed DM that binds to the targetmolecule.

The target molecule may comprise a detectable label in order tofacilitate detection of binding of the target molecule to the displayedpolypeptides. Detectable labels suitable for use in the presentinvention include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, orchemical means. Useful labels in the present invention include biotin orother streptavidin-binding proteins for staining with labeledstreptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescentdyes (e.g., phycoerythrin, YPet, fluorescein, Texas red, rhodamine,green fluorescent protein, and the like, see, e.g., Molecular Probes,Eugene, Oreg., USA), radiolabels (e.g., 3H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P),enzymes (e.g., horse radish peroxidase, alkaline phosphatase and otherscommonly used in an ELISA), and colorimetric labels such as colloidalgold (e.g., gold particles in the 40-80 nm diameter size range scattergreen light with high efficiency) or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads. Patents teaching the useof such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

In some embodiments, the N-terminal, C-terminal, or biterminal fusionCYTX-DPs of the present invention can be used for the identification ofsubstrates, such as protease or kinase substrates, from substratelibraries. Accordingly, a CYTX-DP can be modified to express afluorescent protein using methods known in the art. For example, the useof a bicistronic expression vector comprising a CYTX-DP, (2) a ribosomalbinding site downstream of the CYTX-DP sequence, and (3) a label such asa green fluorescent protein suitable for efficient detection usingfluorescence activated cell sorting (e.g., alajGFP). Expression is thenmonitored through the intensity of green fluorescence. For example, alibrary of protease or peptide substrates is created using methods knownin the art. The substrates are fused to the N-terminus or C-terminus orboth termini of CYTX-DPs using an expression vector expressing a greenfluorescent protein. The substrate library is constructed such that alabel or an affinity tag suitable for fluorescence labeling is fused tothe free terminus of a DM on the cell surface. Host cells expressing thesubstrate library labeled with a red fluorescent protein are grown, andcells that are green but not red are removed from the population toeliminate the isolation of false positive clones. The library is thenincubated with an enzyme (e.g., a protease or peptidase), and cells thatlose red fluorescence while retaining green fluorescence are isolatedfrom the population using FACS.

In some embodiments, the N-terminal, C-terminal, or biterminal fusionCYTX-DPs of the present invention can be used to construct whole cellsthat can be used as reagents. For example, one or more peptidesidentified using the methods herein, binding to a protein, virus, orcellular receptor, or synthetic composition of matter, are displayed onthe outer surface of a bacterial cell at a desired surface density.Cells can then be coupled directly to a material, e.g., glass/silicon,gold, polymer, by virtue of peptides selected to bind these materials,and used to capture in solution molecules binding to various otherdisplayed peptides on the same cell. For optical detection, cells canco-express a fluorescent or luminescent reporter molecule such GFP, orluciferase. Flow cytometry, or fluorescence microscopy can be used todetect binding of molecular recognition element displaying cells to thetarget agent, e.g., virus, cell, particle, bead, and the like.

The polypeptide display systems of the present invention allow thecreation of renewable whole cell binding reagents in non-specializedlaboratories since this method is technically accessible and librariesare reusable. This approach has already proven useful for selectingcell-specific binding peptides, and for performing diagnostic assaysusing flow cytometry and fluorescence microscopy. Furthermore, thesurface displayed polypeptides can be used for parallel or multiplexligand isolation, and clones can be processed with efficient single-celldeposition units present on many cell sorters. See Feldhaus, M. J., etal. (2003) Nat. Biotechnol. 21(2):163-170, which is herein incorporatedby reference. Consequently, the expression vectors of the presentinvention may be used in proteomic applications including proteome-wideligand screens for protein-detecting array development. See Kodadek, T.(2001) Chem. Biol. 8(2):105-115, which is herein incorporated byreference.

In some embodiments, the display systems using CYTX-CP polypeptides ofthe present invention can be used for the identification of substrates,such as protease and peptidase substrates, from substrate libraries.Accordingly, an expression vector may be modified to express afluorescent protein using methods known in the art. For example, the useof a bicistronic expression vector comprising (1) a CYTX-CP polypeptide,(2) a ribosomal binding site downstream of the CYTX-CP nucleic acidsequence, and (3) label such as a green fluorescent protein suitable forefficient detection using fluorescence activated cell sorting, such asalaj GFP. Expression is then monitored through the intensity of greenfluorescence.

A library of the substrates is created using methods known in the art.The substrates are fused to the CYTX-CP expression system, respectively.The substrate library is constructed such that a label or an affinitytag suitable for fluorescence labeling is fused to the free terminus ofthe DM on the cell surface. The library is then grown, and cells thatare green but not red are removed from the population to eliminate theisolation of false positive clones. The library is then incubated withthe enzyme (e.g., a protease or peptidase), and cells that lose redfluorescence while retaining green fluorescence are isolated from thepopulation using FACS.

In some embodiments, the CYTX-CP expression vectors of the presentinvention can be used to construct whole cells that can be used asreagents. For example, one or more peptides identified using the methodsherein, binding to a protein, virus, or cellular receptor, or syntheticcomposition of matter, are displayed on the outer surface of E. coli ata desired surface density. Cells can then be coupled directly to amaterial, e.g., glass/silicon, gold, polymer, by virtue of peptidesselected to bind these materials, and used to capture in solutionmolecules binding to various other displayed peptides on the same cell.For optical detection, cells can co-express a fluorescent or luminescentreporter molecule such GFP, or luciferase. Flow cytometry, orfluorescence microscopy can be used to detect binding of molecularrecognition element displaying cells to the target agent, e.g., virus,cell, particle, bead, and the like.

It should be noted that although the use of bacterial proteins areexemplified herein, a variety of surface localized proteins possessingsurface exposed loops may be modified according to the present inventionto provide expression vectors that allow the display of polypeptides onthe outer surface of viruses, and prokaryotic and eukaryotic cellsincluding phage, bacteria, yeast, and mammalian cells. A variety ofsurface localized proteins known in the art may be used. In Escherichiacoli and substantially similar species, such proteins include OmpA,OmpX, OmpT, OmpC, OmpF, OmpG, OmpN, LamB, FepA, FecA, and otherbeta-barrel outer membrane proteins. Proteins that exhibit a topologysubstantially similar to that shown in FIGS. 6A-6C, i.e., present eithera C- or N-terminus on the outer surface of bacteria, may also be usedaccording to the present invention. One of ordinary skill in the art mayreadily identify and screen for the various surface localized proteinsthat may be used in accordance with the present invention.

The following examples are intended to illustrate but not to limit theinvention.

Example 1 Materials & Methods

Structural Analysis and Design:

The structures of all Outer Membrane Protein (Omp) family members thathave been determined, including structures of OmpX, were analyzed usingPyMol software (Schrodinger, Portland, Oreg. (formerly DeLanoScientific). OmpX structural modifications were developed by visualinspection and by using PyMOL tools and scripts. Molecular biology andengineering of constructs were developed using Vector NTI Suite 11 (LifeTechnologies, Grand Island, N.Y.).

Reagents and Strains:

Oligonucleotides were ordered from Elim Biopharmaceutical (Hayward,Calif.). The vector was modified from pBAD33 including removing andintroducing restriction sites (Boulware, K. T. & Daugherty, P. S.,(2006) PNAS, 103(20), 7583-7588). Molecular biology reagents includingPCR reagents, restriction endonucleases and ligation reagents were fromLucigen (Middleton, Wis.) and New England Biolabs (Ipswich, Mass.).Streptavidin-conjugated phycoerythrin (SA-PE) (Invitrogen) was usedwithout modifications. YPet fused to the SH3 domain of Mona was producedand used as described in U.S. Pat. No. 7,666,817 B2, issued Feb. 23,2010. The N-terminal epitope tag sequence of the CYTX-DP platform was ashort version of the Glu-Glu epitope tag (binds EYMPME (SEQ ID NO: 8),or EFMPME (SEQ ID NO: 43) sequences). The antibody targeting this tagwas the monoclonal mouse IgG1 anti-Glu-Glu (Covance, Princeton, N.J.),also referred to herein as Anti-EE or EE. The C-terminal epitope tagsequence of the CYTX-DP platform was an eight poly-His epitope tag(HHHHHHHH) (SEQ ID NO: 13). The antibody targeting this tag was amonoclonal mouse IgG1 anti-6×His (R&D Systems, Minneapolis, Minn.). Thesecondary antibody targeting hCTLA-4Fc was a monoclonal mouse IgG1 andthe human CTLA-4-Fc was CHO-cell expressed (R&D Systems, Minneapolis,Minn.). Fluorescence labeling kits were AlexaFluor 488 nm and 647 nmlabeling kits from Life Technologies (formerly Invitrogen). For example,anti-EE antibody was labeled with Alexa 647 and designated EE647;anti-His monoclonal antibody was labeled with Alexa 647 and designatedHis647. The conjugated mouse IgG1s were at a stock concentration ofabout 1 mg/ml in PBS and stored at 4 degrees C. E. coli strains usedwere DH-10β from NEB, MC1061 ((Casadaban, M. J. & Cohen, S. N. (1980)JMB, 138, 179-207) and Lucigen), and C41(DE3) and CD43(DE3) (Lucigen andOverExpress, France). Bacteria cultures were grown at 37° C. withvigorous shaking for construct development in Luria-Bertaini (LB) brothsupplemented with 25-35 micrograms per ml (μg/ml) chloramphenicol (CM),unless another antibiotic was specified. Bacterial cultures were grownat 28° C. to 37° C. for protein expression in LB broth with CM (unlessanother antibiotic was specified) and also supplemented with 0.01% to0.04% arabinose for induction. The enzymes human plasmin (HaematologicTechnologies Inc., Essex Junction, Vt.) and recombinant humanurokinase-type Plasminogen Activator (uPA) (Analytical BiologicalServices Inc., Wilmington, Del.) were used without modifications. Humansynovial fluid samples (Analytical Biological Services, Inc.) were usedwithout modifications.

Substrate Cleavage and Platform Stability Analysis:

Susceptibility of eCLiPS platforms (see e.g., FIGS. 20-22, and 26-28)and CYTX-DP platforms (see e.g., FIGS. 23-25, and 29-31) to cleavage byproteases, either specifically at a substrate site or throughoutextracellular portions of the platform, was evaluated as follows.Bacteria transformed by plasmids encoding eCLiPS3.0 or CYTX-DP platformswere grown overnight, subcultured by dilution into fresh media (1:50dilution), and grown for 1.5 to 2 hours. The subculture was then inducedwith 0.04% arabinose and incubated with shaking at 37° C. for 1 hour. Tostop further growth, cells were incubated on ice for 15-30 minutes. Cellaliquots were harvested and washed with PBS (pH 7.4). To test forprotease cleavage susceptibility, cells were pelleted by centrifugation,the supernatant removed, and the cells resuspended in reaction buffercontaining the test enzyme. This reaction mixture was incubated at 37°C. static. To stop the reaction, cells were removed and diluted 10-foldin PBS, pelleted by centrifugation, and resuspended in PBS containingSA-PE (20 μg/mL) or YPet-MONA (50 nM) for eCLiPS3.0 platforms or in PBScontaining EE647 (1 μg/ml) or His 647 (2 ug/ml) for CYTX-DP platforms.After incubation on ice (30 min), cells were washed with PBS andanalyzed using a FACSAria™ cell sorter in a manner similar to thatdescribed in U.S. Pat. No. 7,666,817 B2, ibid.

Assays to measure uPA hydrolysis of CYTX-DP platforms containing uPAsubstrates 1203 or 1204 (i.e., CYTX-DP-1203 or CYTX-DP-1204 platforms,respectively) were performed in TBST (50 mM Tris, 150 mM NaCl, 0.05%Tween20, pH 7.4) with 1 nM-1.25 micromolar (μM) uPA. Such hydrolysis wascompared to that of uPA mixed with eCLiP3.0-1203 or eCLiPS3.0-1204.Background hydrolysis of the regions flanking the substrate site (usingeCLiPs3.0-NSUB and CYTX-DP-NSUB), was measured under each reactioncondition to ensure that hydrolysis occurred in the designated substrateregion. NSUB refers to a non-cleavable substrate. Typically an NSUBcomprises a Glycine-Serine linker of about 1 to about 8 amino acids. Seefor example, SEQ ID NO: 23.

Assays to measure susceptibility of eCLiPS3.0-NSUB or CYTX-DP-NSUBplatforms to human plasmin cleavage were performed in 50 mM Tris-HCl,pH.7.5, supplemented with 100 mM NaCl, 0.01% Tween20, and 1 mM EDTA with100 nM-312.5 μM human plasmin.

Assays to measure susceptibility of eCLiPS3.0-NSUB or CYTX-DP-NSUBplatforms to human synovial fluid were performed with synovial fluidsamples in 10%-90% PBS, pH 7.4.

Amino- and Carboxy-terminus Labeling Conditions for eCLiPS3.0:

Streptavidin conjugated phycoerythrin (SA-PE) was used for labelingstreptavidin binding affinity ligand on the N-termini of eCLiPS3.0.Fluorescent protein YPet fused to the SH3 domain of Mona was used forlabeling the MONA binding affinity ligand on the C-termini of eCLiPS3.0.For optimum labeling of cells without protease reaction, the cells wereincubated for 30 min at 4° C. with SA-PE (20 μg/mL) or YPet-MONA (50nM).

Amino- and Carboxy-terminus Labeling Conditions for CYTX-DP:

Alexa-647 conjugated anti-EE antibody (EE647) was used for labeling EEbinding affinity ligand on the N-termini of CYTX-DP. Alexa-647conjugated anti-His antibody (His647) was used for labeling the 8-Hisbinding affinity ligand on the C-termini of CYTX-DP. For optimumlabeling of cells without protease reaction, the cells were incubatedfor 30 min at 4° C. with Alexa-647 (1 μg/mL) or His647 (2 μg/ml).

Kinetic Data Analysis:

The extent of conversion of cell surface displayed peptide substrateswas measured directly, using flow cytometry to measure changes in meanfluorescence of clonal cell populations upon protease treatment.Specifically, for each sample, conversion was determined by flowcytometry analyses using the relationship

$\begin{matrix}{{Conversion}_{CLiPS} = \frac{{{FL}\;\_} - {FL}_{+}}{{{FL}\;\_} - {FL}_{0}}} & \lbrack 1\rbrack\end{matrix}$where (FL⁻) is the fluorescence after incubating without enzyme, (FL₊)is fluorescence after incubation with enzyme, and (FL₀) is fluorescenceof unlabeled cells. Given that the expected substrate concentrationsthat were used are significantly below the expected K_(M) of thesubstrate for the target protease, the Michaelis-Menton model simplifiesto

$\begin{matrix}{\frac{\mathbb{d}\lbrack S\rbrack}{\mathbb{d}t} \approx {- {{\frac{k_{\;{cat}}}{k_{M}}\lbrack S\rbrack}\lbrack E\rbrack}}} & \lbrack 2\rbrack\end{matrix}$allowing substrate conversion to be expressed as

$\begin{matrix}{{Conversion}_{MM} = {1 - {\exp\left( {{- {\frac{k_{cat}}{k_{M}}\lbrack E\rbrack}} \cdot t} \right)}}} & \lbrack 3\rbrack\end{matrix}$where [S] is the substrate concentration, [E] is enzyme concentrationand t is time. To determine the second order rate constant(k_(cat)/K_(M)), the time dependent conversion for each substrate wasfit to equation [3].

Binding Screening:

For expression screening and clone analysis, cultures were grown intolog-phase or near saturation (>8 hours) and sub-cultured by dilutioninto fresh medium (range from 1/50 to 1/15 dilution) and grownover-night at 28 to 37 degrees C. These cultures were induced with 0.01to 0.04% arabinose during this overnight subculture. The optical densityat 600 nm (OD600) was measured to estimate cell concentration in orderto normalize the cultures to the same cell density after the cultureswere chilled on ice for 30-60 minutes. A calculation per labelingexperiment volume required a maximum of OD600=0.5 for 50 uL cells for a50 uL labeling reaction; when smaller labeling volumes were used, thenfewer cells were used maintaining the equivalent of OD60=0.5 for 50 uLcells for a 50 uL labeling reaction. Cells were washed in 1 mL 1×PBS or1 ml 1×PBS supplemented with 1% BSA and were pelleted by centrifugationat 3500×g for 5 minutes at 4 degrees C. Subsequently, washed cells werelabeled in 1×PBS or 1×PBS supplemented with 1% BSA for 30-60 minutes onice in the dark. Cells were pelleted by centrifugation at 3500×g for 5minutes at 4 degrees C. Cells were re-suspended in 1×PBS 0.3-0.5 mL andanalyzed using a FACSAria custom-ordered sorter (Becton Dickinson,Franklin Lakes, N.J.).

Example 2 Strategy for Producing a More Protease Resistant DisplayPlatform

This Example provides the strategy for producing a display platform forpeptides that is less susceptible to protease cleavage.

Since CLiPS (see e.g., PCT Publication No. WO 2005/047461; PCTPublication No. WO 2009/014726; U.S. Pat. No. 7,256,038; U.S. Pat. No.7,612,019; U.S. Patent Application Publication No. 2010/0113303; PCTPublication No. WO 2007/027935; U.S. Pat. No. 7,666,817; U.S. PatentApplication Publication No. 20100173349, each of which is herebyincorporated by reference in its entirety) is used to isolate novelprotease substrates, it was desirable to create a more robust displayplatform with improved properties of stability toward target proteasesthat were known to digest the platform and toward complex mixtures withhigh protease activity (e.g., synovial fluids). This improved platform,CYTX-DP, allows for more effective protease substrate libraries, masklibraries and, surprisingly, scFv engineering for antibody, protein,probody and pro-protein discovery due to overall stability andengineering of the extracellular domains.

Since the CLiPS platform had been notably susceptible to proteasedegradation outside the substrate library site, the structure and aminoacid sequences of OmpX and CLiPS was analyzed for sites to decrease theprotease liability. Structural analysis highlighted Arg, Lys and Aspamino acid residues on the OmpX loops 2 and 3 that were likely above thelipopolysaccharides (LPS) of the outer membrane and therefore exposed toproteases with specificity to those residues (FIG. 6B, 6C). Inspectionand overlay of the Omp structures in the protein database (RCSB ProteinData Bank, available online) provided additional anchor points abovewhich the Omp/CLiPS transmembrane domain would be flexible and exposedto proteases (FIG. 7); this analysis also considered liabilities.Additionally, amino acid sequence analysis showed similar amino acidresidues in the N-terminal and C-terminal tags.

These analyses were used to modify the CLiPS platform using standardmolecular biology techniques. The fragments of OmpX loop 2, the CLiPSresidues/structure leading from and to the C- and N-terminal tags andsubstrates, were analyzed by site-directed mutagenesis of the respectiveamino acids to alanine residues for decreased labeling on the bacterialsurface (FIG. 8). The fragments of OmpX loop 2, the CLiPSresidues/structure leading from and to the C- and N-terminal tags wereconverted to flexible GlyGlySer (GGS) repeat linkers (so-called “GS”flexible linkers, also referred to herein as GS linkers) compatible withthe length of the replaced structures leading into the membrane buriedbeta-sheet structures. The GS linkers allowed the epitope tags to belabeled and also to be stable toward many proteases due to the nature ofglycine-rich sequences being less protease sensitive. The OmpX loop 3was also considered a protease liability. It was truncated, whileretaining residues for the beta-sheet structure in the membrane; glycineresidues and a proline residue were incorporated to stabilize thehairpin turn.

Example 3 Production of Improved Display Platforms

This Example describes the production of display platforms from whichprotease-labile amino acid sequences have been removed. Such displayplatforms are called CYTX-DP display platforms.

The plasmid comprising eCLiPS3.0-GSNS, the display platform from whichCYTX-DP-GSNS, which includes an EagI restriction site at the C-terminusprior to the histidine tag and is referred to herein as “CYTX-DP-GSNS,”was produced is designated pB33-GSNS1-GGS4-eCLiPS3.0 PHB4901; the aminoacid sequence of eCLIPS3.0-GSNS is shown in FIG. 10 (designatedN-termTag_stop_CLiPS_GSNS). This is a CLiPS construct with the legumain“GSNS” substrate (SEQ ID NO: 10) and a SA-PE N-terminal tag and a YPETC-terminal tag; this YPET tag is designated BV99 and has amino acidsequence HISQWKPKVPNREDKYKK (SEQ ID NO: 18). After protease-labileregions of eCLiPS3.0-GSNS were identified for mutagenesis, standardmolecular biology techniques were used to mutate and truncate thatplatform to make the CYTX-DP-GSNS display platform (i.e., a CYTX-DPplatform comprising a legumain GSNS substrate). The schematic diagram inFIG. 9 shows the signal peptide of eCLiPS3.0 was kept the same alongwith the stop codon. The N- and C-terminal tags of CYTX-DP are smallerand different than those of eCLiPS3.0: CYTX-DP has an N-terminal Glu-Glu(EE) tag with the sequence, EYMPME (SEQ ID NO: 8) and a C-terminal 8-Histag with the sequence, HHHHHHHH (SEQ ID NO: 13).

The amino acid sequence of the CYTX-DP-GSNS platform, which includes asignal peptide (underlined) (SEQ ID NO: 6)—Linker 1 (L1) (SEQ ID NO:7)—EE tag (SEQ ID NO: 8)—Linker 3 (L3) (SEQ ID NO: 9 SEQ ID NO: 59)—GSNSSubstrate (SEQ ID NO: 10)—Linker 3 (L3) (SEQ ID NO: 11)—transmembraneregion (i.e., mutated circularly permuted outer membrane protein) (SEQID NO: 1)—Linker 4 (L4) (SEQ ID NO: 83)—His tag (SEQ ID NO: 13)—stopcodon (*), where brackets indicate the location of each of the elements,is:

(SEQ ID NO: 2) [MKKIACLSALAAVLAFTAGTSVA][GQSGQ][EYMPME][GGSGQSGQGS][GSNS][GSSGGQGGSGGSGGSGGSGGSA][YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYT][GGSGGSSGQTAAG][HHHHHHHH][*]

This CYTX-DP-GSNS platform is encoded by the following nucleic acidsequence:

(SEQ ID NO: 3) atgaaaaaaattgcatgtctttcagcactggccgcagttctggctttcaccgcaggtacttccgtagctggtcaatctggacaggaatacatgccgatggaaggagggtctggccagtctggccagggttctggcagcaattccggttctagcggtggccagggtggcagcggtggctctggtggttccggtggctctggtggctctgcgtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggttctggcccgggtggttcttacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattctgtccgttggttaccgcttcggctccaaatcccgccgtgcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccggcggctctggtggttctagcggtcaaacggccgctggtcaccatcaccaccatcatcaccactaa

Another suitable CYTX-DP platform is a variant of the CYTX-DP-GSNSplatform, where the EagI restriction site is replaced with a NotIrestrictions site. This version, referred to herein as “CYTX-DP-GSNS(NotI)” includes a signal peptide (underlined) (SEQ ID NO: 6)—Linker 1(L1) (SEQ ID NO: 7)—EE tag (SEQ ID NO: 8)—Linker 3 (L3) (SEQ ID NO:59)—GSNS Substrate (SEQ ID NO: 10)—Linker 3 (L3) (SEQ ID NO:11)—transmembrane region (i.e., mutated circularly permuted outermembrane protein) (SEQ ID NO: 1)—Linker 4 (L4) (SEQ ID NO: 12)—His tag(SEQ ID NO: 13)—stop codon (*), where brackets indicate the location ofeach of the elements, is:

(SEQ ID NO: 60) [MKKIACLSALAAVLAFTAGTSVA][GQSGQ][EYMPME][GGSGQSGQGS][GSNS][GSSGGQGGSGGSGGSGGSGGSA][YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYT][GGSGGSSGQAAAG][HHHHHHHH][*]

The mature CYTX-DP-GSNS platform (i.e., lacking the signal sequence) hasthe following amino acid sequence (Linker 1 (L1) (SEQ ID NO: 7)—EE tag(SEQ ID NO: 8)—Linker 3 (L3) (SEQ ID NO: 59)—GSNS Substrate (SEQ ID NO:10)—Linker 3 (L3) (SEQ ID NO: 11)—transmembrane region (i.e., mutatedcircularly permuted outer membrane protein) (SEQ ID NO: 1)—Linker 4 (L4)(SEQ ID NO: 83)—His tag (SEQ ID NO: 13)—stop codon (*), where bracketsindicate the location of each of the elements):

(SEQ ID NO: 4) [GQSGQ][EYMPME][GGSGQSGQGS][GSNS][GSSGGQGGSGGSGGSGGSGGSA][YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYT][GGSGGSS GQTAAG][HHHHHHHH][*]

The mature CYTX-DP-GSNS platform is encoded by the following nucleicacid sequence:

(SEQ ID NO: 5) ggtcaatctggacaggaatacatgccgatggaaggagggtctggccagtctggccagggttctggcagcaattccggttctagcggtggccagggtggcagcggtggctctggtggttccggtggctctggtggctctgcgtactacggcatcactgctggtccggcttaccgcattaacgactgggcaagcatctacggtgtagtgggtgtgggttatggttctggcccgggtggttcttacggtttctcctacggtgcgggtctgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattctgtccgttggttaccgcttcggctccaaatcccgccgtgcgacttctactgtaactggcggttacgcacagagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctgggtgtgatcggttctttcacttacaccggcggctctggtggttctagcggtcaaacggccgctggtcaccatcaccaccatcatcac  cactaa

Another suitable mature CYTX-DP platform is a variant of the matureCYTX-DP-GSNS platform, where the EagI restriction site is replaced witha NotI restrictions site. This version, referred to herein as “matureCYTX-DP-GSNS (NotI)” includes Linker 1 (L1) (SEQ ID NO: 7)—EE tag (SEQID NO: 8)—Linker 3 (L3) (SEQ ID NO: 59)—GSNS Substrate (SEQ ID NO:10)—Linker 3 (L3) (SEQ ID NO: 11)—transmembrane region (i.e., mutatedcircularly permuted outer membrane protein) (SEQ ID NO: 1)—Linker 4 (L4)(SEQ ID NO: 59 SEQ ID NO: 12)—His tag (SEQ ID NO: 13)—stop codon (*),where brackets indicate the location of each of the elements, is:

(SEQ ID NO: 58) [GQSGQ][EYMPME][GGSGQSGQGS][GSNS][GSSGGQGGSGGSGGSGGSGGSA][YYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYT][GGSGGSS GQAAAG][HHHHHHHH][*]

FIG. 10 demonstrates alignment of nucleic acid sequences encoding thedescribed CYTX-DP-GSNS display platform (upper rows) and eCLiPS3.0-GSNSdisplay platform (lower rows). The two nucleic acid sequences are 74.7%identical. FIG. 11 demonstrates alignment of the amino acid sequences ofthe described CYTX-DP-GSNS display platform (upper rows) andeCLiPS3.0-GSNS display platform (lower rows). The two amino acidsequences are 71.2% identical.

FIGS. 23-25 and FIGS. 29-31 are schematic representations of the aminoacid sequence and structural arrangement of various embodiments ofdisplay platforms according to the present invention.

In some embodiments, the nucleotide sequence encoding the C-terminus ofCYTX-CP is modified at the EagI/NotI restriction site before thenucleotide sequence encoding the 8×His tag to substitute a nucleotidesequence encoding one of the following C-termini incorporating the Histag: EagI variant (TAAGHHHHHHHH*) (SEQ ID NO: 53), or NotI variant(AAAGHHHHHHHH*) (SEQ ID NO: 54). In one embodiment a nucleotide sequenceencoding a TLA-TAIL is substituted into a nucleotide sequence encoding ascFv-containing DP at the NotI restriction site near the C-terminus toencode a TLA-TAIL His tag (AAAGEIVLTQSPGTLVTVSSHHHHHHHH*) (SEQ ID NO:55); such a tag can lead to enhanced labeling.

Example 4 Characterization of Substrate Cleavage Kinetics in CLiPS andCYTX-DP Scaffolds

This Example compares the ability of a protease to cleave its substratein CYTX-DP display platform versus in a CLiPS display platform.

Cultures of clones, each comprising a plasmid encoding CYTX-DP-1203(i.e., a CYTX-DP platform comprising substrate 1203, depicted in FIG.30), CYTX-DP-1204 (depicted in FIG. 31), eCLiPS3.0-1203 (depicted inFIG. 27), or eCLiPS3.0-1204 (depicted in FIG. 28), respectively, weregrown and exposed to uPA protease under the conditions described in theExamples herein. Relative cleavage kinetics of the substrates in thevarious platforms was assessed using flow cytometry. The individualclones exhibited uniform substrate turnover, as determined by flowcytometry. In this way, the extent of conversion for each clone could bedetermined at several different protease concentrations and fit to aMichaelis-Menton model (see Kinetic Data Analysis section in Example 1).FIG. 13 demonstrates that the observed second order rate constant(k_(cat)/K_(M)) for each substrate was comparable across both the CLiPSand CYTX-DP platforms. Background hydrolysis of the regions flanking thesubstrate site (using eCLiPS3.0-NSUB (depicted in FIG. 26) andCYTX-DP-NSUB (depicted in FIG. 29)), was measured under each reactioncondition to ensure that hydrolysis occurred in the designated substrateregion.

Example 5 Protease Resistance of the CLiPS and CYTX-DP Display Platforms

This Example compares the stability of the CYTX-DP and CLiPS displayplatforms in the presence of the protease plasmin.

Cultures of clones, each comprising a plasmid encoding CYTX-DP-NSUB oreCLiPS3.0-NSUB, were grown and exposed to plasmin under the conditionsdescribed in the Examples herein. Loss of either the N-terminal orC-terminal affinity tag from either display platform resulted in areduction of the mean fluorescence intensity, which was assessed by flowcytometry. Conversion of each platform to a platform lacking either theN-terminal or C-terminal affinity tag was then determined (see KineticData Analysis section herein). Loss of either affinity tag due toprotease cleavage in the non-substrate region would be detrimental tothe ability to screen libraries in the presence of human plasmin. Boththe N-terminal and C-terminal affinity tags of CYTX-DP showed increasedresistance over CLiPS in the presence of human plasmin, as shown inFIGS. 14A, 14B. The significantly greater stabilities exhibited byCYTX-DP over CLiPS improves the ability to select substrates fromCYTX-DP substrate libraries. In addition, the 52-fold greater stabilityof the N-terminus in CYTX-DP allows the characterization of substrateresistance against undesirable proteases (e.g., proteases in serum or atan off-target site) at substantially higher protease concentrations.

Example 6 Improved Resistance of the CYTX-DP Display Platform in thePresence of Synovial Fluid

This Example shows the improved stability of CYTX-DP against a complexmixture expected to contain a spectrum of proteases.

Cultures of clones, each comprising a plasmid encoding CYTX-DP-NSUB oreCLiPS3.0-NSUB, were grown and exposed to human synovial fluid samplesunder the conditions described in the Examples herein. Synovial fluidwas expected to have a wide range of protease species at varyingconcentrations. Loss of either the N-terminal or C-terminal affinity tagfrom either display platform resulted in a reduction of the meanfluorescence intensity, which was assessed by flow cytometry. Loss ofeither affinity tag due to protease cleavage in the non-substrate regionwould be detrimental to the ability to screen libraries in the presenceof synovial fluid. FIG. 15 demonstrates that the N-terminal affinity tagof CYTX-DP shows increased resistance over that of CLiPS in the presenceof synovial fluid during a 1 hr incubation at 37° C.

Example 7 Characterization of scFv Expressed at the C-Terminus ofCYTX-DP

The use of a multi-copy display on whole cells enabled simple and directquantitation of expression on the bacterial cell surface. Plasmidsencoding scFv F5 or scFv OKT3 fused to CYTX-DP at the C-terminus beforethe C-terminal 8×His epitope tag (referred to as display platformCYTX-DP-scFvFS-Cterm, or CYTX-DP-scFvOKT3-Cterm, respectively) wereexpressed in E. coli DH-10β overnight with induction started from a1/20-1/50 dilution of a nearly saturated culture. The sequences for theF5 scFv and OKT3 scFv are shown below:

F5 scFv amino acid sequence: (SEQ ID NO: 44)QVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAE QKLISEEDLNGAA OKT3 scFv amino acid sequence: (SEQ ID NO: 45)QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSI KLISEEDLNGAA 

The following CYTX-DP platforms were used in the methods describedherein. In some embodiments, the CYTX-DP platform includes a signalpeptide (“SS”). In some embodiments, the CYTX-DP platform is a matureversion, which does not include a signal peptide. These embodiments arereferred to herein as “mature CYTX-Dplatforms.”

In some embodiments, the scFv was expressed at the C-terminus of theCYTX-DP platform. These embodiments are referred to herein as“C-terminal CYTX-DP Platforms” and include the following structuralarrangement from N-terminus to C-terminus: SS-T1-L1-CP-scFv-L2-T2.

In some embodiments, the scFv was expressed at the N-terminus of theCYTX-DP platform. These embodiments are referred to herein as“N-terminal CYTX-DP Platforms” and include the following structuralarrangement from N-terminus to C-terminus: (SS-T1-L1-scFv-CP-L2-T2). Insome embodiments, the scFv was expressed at the N-terminus of theCYTX-DP platform, and the C-terminus included a tail sequence (“TAIL”).These embodiments are referred to herein as “N-terminal scFv, C-terminalTail CYTX-DPlatforms” and include the following structural arrangementfrom N-terminus to C-terminus: (SS-T1-L1-scFv-CP-L2-TAIL-T2).

C-terminal CYTX-DP Platforms with Signal Sequence(SS-T1-L1-CP-scFv-L2-T2): F5 C-terminal CYTX-DP platform (SS-T1-L1-CP-F5scFv-L2-T2) (SEQ ID NO: 62)MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGQGSGSNSGSSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGQVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAEQKLISEEDLNGAAHHHHHH*  OKT3 C-terminal CYTX-DP platform (SS-T1-L1-CP-OKT3scFv-L2-T2) (SEQ ID NO: 63)MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGQGSGSNSGSSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIKLISEEDLNGAAHHHHHH* N-terminal CYTX-DP Platforms with Signal Sequence(SS-T1-L1-scFv-CP-L2-T2): F5 N-terminal CYTX-DP platform (SS-T1-L1-F5scFv-CP-L2-T2) (SEQ ID NO: 64)MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGQVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAEQKLISEEDLNGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHHH* OKT3 N-terminal CYTX-DP platform (SS-T1-L1-OKT3scFv-CP-L2-T2) (SEQ ID NO: 65)MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIKLISEEDLNGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHHH*  N-terminal scFv, C-terminal Tail CYTX-DPPlatforms with Signal Sequence  (SS-T1-L1-scFv-CP-L2-TAIL-T2):F5 N-terminal scFv, C-terminal Tail CYTX-DPplatform (SS-T1-L1-F5scFv-CP-L2-TAIL-T2) (SEQ ID NO: 66)MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGQVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAEQKLISEEDLNGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGEIVLTQSPGTLVTVSSHHHHHHHH*Mature C-terminal CYTX-DP Platforms  (T1-L1-CP-scFv-L2-T2):Mature F5 C-terminal CYTX-DP platform  (T1-L1-CP-F5scFv-L2-T2)(SEQ ID NO: 67) GQSGQEYMPMEGGSGQSGQGSGSNSGSSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGQVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAEQKLISEEDLNG AAHHHHHH* Mature OKT3 C-terminal CYTX-DP platform  (T1-L1-CP-OKT3scFv-L2-T2)(SEQ ID NO: 68) GQSGQEYMPMEGGSGQSGQGSGSNSGSSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIKLISEEDLNGA AHHHHHH* Mature N-terminal CYTX-DP Platforms  (T1-L1-scFv-CP-L2-T2):Mature F5 N-terminal CYTX-DP platform  (T1-L1-F5scFv-CP-L2-T2)(SEQ ID NO: 69) GQSGQEYMPMEGGSGQSGGQVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAEQKLISEEDLNGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHH H*Mature OKT3 N-terminal CYTX-DP platform (T1-L1-OKT3scFv-CP-L2-T2)(SEQ ID NO: 70) GQSGQEYMPMEGGSGQSGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIKLISEEDLNGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHHH*Mature N-terminal scFv, C-terminal Tail CYTX-DPPlatforms (T1-L1-scFv-CP-L2-TAIL-T2):Mature N-terminal scFv, C-terminal Tail F5 CYTX-DP platform (T1-L1-F5scFv-CP-L2-T2) (SEQ ID NO: 71)GQSGQEYMPMEGGSGQSGGQVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISYTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGNTNRPSQVPDRFSGFKSGTSASLAITGLQAEDEADYYCQSYDSSLSQWVFGGGTKLTVLGAAAEQKLISEEDLNGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGEIVLTQSP GTLVTVSSHHHHHHHH* 

Expression was induced with 0.01% arabinose or suppressed with 0.2%glucose for >12 hours at 28° C. with vigorous shaking after which cellswere put on ice for 1 hour. The OD600 for each culture was measured andsamples were normalized for cells to OD600 of 0.5 for 50 uL of cells perlabeling or binding experiment. Cells were washed with 1 ml PBS(phosphates buffered saline) at 4° C. and centrifuged at 3500×g for 5min at 4° C. Labeling was done at 1/500 N-terminal epitope tag label(anti-EE 647 label) or 1/100 C-terminal epitope tag label (anti-poly-His488 label) with 0.1% BSA for 1 hour on ice. Cells were centrifuged at3500×g for 5 minutes and supernatant aspirated. Prior to flow cytometry(FACSAria) analysis, cells were re-suspended in 0.3 to 0.5 mL ice coldPBS. Based on unlabeled cells being in the lower left quadrant on alog-scale, cells were determined to be expressing scFv and labeling whenshifted above the ‘dark’ population by 1-2 log shift in fluorescenceintensity. The suppressed cell population had slightly differentcharacteristics due to the changes in cell populations shape and size,where gating for both the suppressed and induced population improvedselection of labeled populations. Overall, both CYTX-DP-scFvFS-Cterm andCYTX-DP-scFvOKT3-Cterm expressed at levels from 2.8- to 9-fold overun-induced or suppressed cells, when labeled with the tags at either theN- or C-terminus, as shown in FIG. 16.

Example 8 Characterization of scFv Expressed on the N- or C-Terminus ofCYTX-DP

The use of a multi-copy display on whole cells enabled simple and directquantitation of expression on the bacterial cell surface. Plasmidsencoding scFv F5 or scFv OKT3 fused to CYTX-DP at either the C-terminusbefore the C-terminal 8×His epitope tag (CYTX-DP-scFvF5-Cterm, orCYTX-DP-scFvOKT3-Cterm, respectively) or at the N-terminus after theN-terminal EE epitope tag (CYTX-DP-scFvF5-Nterm orCYTX-DP-scFvOKT3-Nterm, respectively) were expressed in E. coli DH-10βovernight with induction started from a 1/20-1/50 dilution of a nearlysaturated culture. Expression was induced with 0.01% arabinose orsuppressed with 0.2% glucose for >12 hours at 28° C. with vigorousshaking after which cells were put on ice for 1 hour. The OD600 for eachculture was measured and samples were normalized for cells to OD600 of0.5 for 50 uL of cells per labeling or binding experiment. Cells werewashed with 1 ml PBS (phosphates buffered saline) at 4° C. andcentrifuged at 3500×g for 5 min at 4° C. Labeling was done at 1/500N-terminal epitope tag label (anti-EE 647 label) with 0.1% BSA for 1hour on ice. Cells were centrifuged at 3500×g for 5 minutes andsupernatant aspirated. Prior to flow cytometry (FACSAria) analysis,cells were re-suspended in 0.3 to 0.5 mL ice cold PBS. Based onunlabeled cells being in the lower left quadrant on a bi-log-scale dotplot, cells were determined to be expressing scFv and labeling at theN-terminal EE epitope tag with anti-EE mIgG1-AlexaFluor647 when shiftedabove the ‘dark’ population by 1-2 log shift in fluorescence intensity(arbitrary units). The suppressed cell population had slightly differentcharacteristics due to the changes in cell populations' shape and size,where gating for both the suppressed and induced population improvedselection of labeled populations. FIG. 17 demonstrates that F5 scFvexpressed and labeled 4-fold over un-induced cells as the N-terminalfusion with CYTX-DP(CYTX-DP-scFvFS-Nterm) and 14-fold over un-induced asthe C-terminal fusion with CYTX-DP(CYTX-DP-scFvFS-Cterm); OKT3 scFvexpressed 8-fold over un-induced cells for both the N-terminal andC-terminal fusions with CYTX-DP (CYTX-DP-scFvOKT3-Nterm andCYTX-DP-scFvOKT3-Cterm, respectively). In summary, scFvs expressed asfusions with CYTX-DP at either the N-terminus or the C-terminus andlabeled at the N-terminal epitope tag.

Example 9 Characterization of scFv Expression and Antigen Binding whenscFv is Fused at the N-Terminus of CYTX-DPs

The use of a multi-copy display on whole cells enabled simple and directquantitation of expression and antigen binding on the bacterial cellsurface. Plasmids encoding scFv F5 or anti-CTLA-4 clone 2 fused toCYTX-DP at the N-terminus after the N-terminal EE epitope tag displayplatforms CYTX-DP-scFvF5-Nterm or CYTX-DP-antiCTLA4-Nterm,respectively). were expressed in E. coli DH-10β or E. coli C41(DE3)overnight with induction started from a 1/20 dilution of a nearlysaturated culture. The sequence for the F5 scFv is shown above in SEQ IDNO: 44. The sequence for the anti-CTLA-4 clone 2 scFv is shown below:

Anti-CTLA-4 clone 2 scFv amino acid sequence: (SEQ ID NO: 46)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVT  VSSAS

An additional scFv sequence characterized using these methods includesthe following anti-gp130 scFv sequence:

Anti-gp130 amino acid Sequence (Hv-L-Lv) (SEQ ID NO: 72)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSRGQNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIISTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQETMPPTFGQGTKVEIKR 

The following CYTX-DP platforms were used in the methods describedherein. In some embodiments, the CYTX-DP platform includes a signalpeptide (“SS”). In some embodiments, the CYTX-DP platform is a matureversion, which does not include a signal peptide. These embodiments arereferred to herein as “mature CYTX-Dplatforms.”

In some embodiments, the scFv was expressed at the N-terminus of theCYTX-DP platform. These embodiments are referred to herein as“N-terminal CYTX-DP Platforms” and include the following structuralarrangement from N-terminus to C-terminus: (SS-T1-L1-scFv-CP-L2-T2). Insome embodiments, the scFv was expressed at the N-terminus of theCYTX-DP platform, and the C-terminus included a tail sequence (“TAIL”).These embodiments are referred to herein as “N-terminal scFv, C-terminalTail CYTX-DPlatforms” and include the following structural arrangementfrom N-terminus to C-terminus: (SS-T1-L1-scFv-CP-L2-TAIL-T2).

N-terminal CYTX-DP Platforms with Signal Sequence (SS-T1-L1-scFv-CP-L2-T2):anti-CTLA-4 N-terminal CYTX-DP platform (SS-T1-L1- CTLA-4scFv-CP-L2-T2)(SEQ ID NO: 73) MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRATLVTVSSASGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSG QTAAGHHHHHHHH*anti-gp130 N-terminal CYTX-DP platform (SS-T1-L1- gp130scFv-CP-L2-T2)(SEQ ID NO: 74) MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSRGQNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIISTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQETMPPTFGQGTKVEIKRGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHH HHH*N-terminal scFv, C-terminal Tail CYTX-DP Platforms with Signal Sequence (SS-T1-L1-scFv-CP-L2-TAIL- T2):anti-CTLA-4 CYTX-DP platform (SS-T1-L1-CTLA-4scFv- CP-L2-TAIL-T2)(SEQ ID NO: 75) MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRATLVTVSSASGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGEIVLTQSPGTLVTVSSHHHHHHHH*anti-gp130 CYTX-DP platform (SS-T1-L1-gp130scFv-CP- L2-TAIL-T2)(SEQ ID NO: 76) MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSRGQNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIISTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQETMPPTFGQGTKVEIKRGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGEIVLT QSPGTLVTVSSHHHHHHHH*Mature N-terminal CYTX-DP Platforms (T1-L1-scFv- CP-L2-T2):Mature anti-CTLA-4 N-terminal CYTX-DP platform (T1-L1-CTLA-4scFv-CP-L2-T2) (SEQ ID NO: 77)GQSGQEYMPMEGGSGQSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRATLVTVSSASGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHHH*Mature anti-gp130 N-terminal CYTX-DP platform (T1-L1-gp130scFv-CP-L2-T2) (SEQ ID NO: 78)GQSGQEYMPMEGGSGQSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSRGQNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIISTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQETMPPTFGQGTKVEIKRGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHHH*Mature N-terminal scFv, C-terminal Tail CYTX-DP Platforms (T1-L1-scFv-CP-L2-TAIL-T2):Mature anti-CTLA-4 CYTX-DP platform (T1-L1-CTLA- 4scFv-CP-L2-TAIL-T2)(SEQ ID NO: 79) GQSGQEYMPMEGGSGQSGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRATLVTVSSASGSGGQGGSGGSGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGEIVLTQSPGTLVTVSSHHH HHHHH*Mature anti-gp130 CYTX-DP platform (SS-T1-L1- gp130scFv-CP-L2-TAIL-T2)(SEQ ID NO: 80) GQSGQEYMPMEGGSGQSGGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSRGQNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIISTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQETMPPTFGQGTKVEIKRGSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGEIVLTQSPGTLVTVSSHHHHHHHH*

Expression was induced with supplementing the culture with 0.01%arabinose for >12 hours at 28° C. or 37° C. with vigorous shaking afterwhich cells were put on ice for 1 hour. The OD600 for each culture wasmeasured and samples were normalized for cells to OD600 of 0.5 for 50 uLof cells per labeling or binding experiment. Cells were washed with 1 mlPBS (phosphates buffered saline) supplemented with 1% BSA at 4° C. andcentrifuged at 3500×g for 5 min at 4° C. To measure expression, cellswere labeled for 1 hour on ice in PBS supplemented with 1% BSA with theN-terminal EE epitope mIgG1 conjugated with AlexaFluor647 at 1/100dilution (the APC channel y-axis in panels A-D of FIG. 18). To measureability of the display platforms to bind CTLA-4 antigen, cells wereincubated for 1 hour on ice in PBS supplemented with 1% BSA with humanCTLA-4Fc at 0.06 mg/ml along with 1/200 anti-human Fc IgG-AlexaFluor488.The antibodies in display platforms CYTX-DP-scFvFS-Nterm andCYTX-DP-antiCTLA4-Nterm expressed at the percentages shown in the APCchannel y-axis, FIG. 18, Panels A-D, upper left quadrant, gate P3. Theantibodies in CYTX-DP-scFvFS-Nterm and CYTX-DP-antiCTLA4-Nterm expressedat 16% (the FITC channel x-axis, FIG. 18, Panels A, B, gate P3) as aheterogeneous population in DH-103 at 28 degrees C. The antibodies inCYTX-DP-antiCTLA4-Nterm expressed at 6.4% with a separate population inthe upper left quadrant (FIG. 18, Panel C, gate P3) in C41(DE3) at 28degrees. The anti-CTLA4 in CYTX-DP-antiCTLA4-Nterm expressed at 11% witha separate population in the upper left quadrant (FIG. 18, Panel D, gateP3) in C41(DE3) at 37 degrees. For CTLA-4 antigen binding, theantibodies in CYTX-DP-scFvFS-Nterm and CYTX-DP-antiCTLA4-Nterm expressedon DH-10β at 28° C. bound human CTLA-4-Fc at less than 1% (FIG. 18,Panels E, F gate Antigen binding). For CTLA-4 antigen binding, theanti-CTLA-4 in CYTX-DP-antiCTLA4-Nterm expressed on C41(DE3) at 28 and37° C. bound human CTLA-4-Fc at less than 1% and 2.5%, respectively(FIG. 18, Panel G, H, gate Antigen binding). Anti-CTLA-4 clone 2antibody appears to display as a functional antibody on E. coli C41(DE3)cells in view of the antibody's ability to bind CTLA-4 antigen.Expression of the anti-gp130 antibody scFv in C43(DE3) E. coli. Panel Ishows N-terminal labeling with anti-EE epitope tag antibody conjugatedwith Alex488. Six percent of the population is expressing the anti-EEepitope tag. Panel J shows that the anti-gp130 scFv expressing bacteriabind biotinylated soluble, human gp130 and are labeled with secondarystreptavidin-PE (SAPE) at 1/50 dilution. 2.7% of the population bindssoluble gp130.

Example 10 Comparison of scFv Expression in CLiPS and CYTX-DP Platformsin Different Cell Types

The use of a multi-copy display on whole cells enabled simple and directquantitation of expression and antigen binding on the bacterial cellsurface. Plasmids encoding scFv F5 or anti-CTLA-4 clone 2 fused toeither eCLiPS3.0 or CYTX-DP at the N-terminus after the N-terminal SAPE(in CLiPS) or EE epitope tag (in CYTX-DP) (i.e., eCLiPS3.0-scFvF5-Nterm,eCLiPS3.0-antiCTLA4-Nterm, CYTX-DP-scFvF5-Nterm orCYTX-DP-antiCTLA4-Nterm) were expressed in E. coli DH-10β, E. coliC41(DE3), or E. coli C43(DE3) (a C41(DE3) variant) overnight withinduction started from a 1/20 dilution of a nearly saturated culture, asindicated in FIG. 19. Expression was induced by supplementing theculture with 0.01% arabinose for >12 hours at 28° C. with vigorousshaking after which cells were put on ice for 1 hour. The OD600 for eachculture was measured and samples were normalized for cells to OD600 of0.5 for 50 uL of cells per labeling experiment. Cells were washed with 1ml PBS (phosphates buffered saline) supplemented with 1% BSA at 4° C.and centrifuged at 3500×g for 5 min at 4 degrees C. To measureexpression, cells were labeled for 1 hour on ice in PBS supplementedwith 1% BSA with the N-terminal epitope tag label SAPE at 1/150 dilutionfor CLiPS (the PE channel x-axis in FIG. 19, panels A and C) or mIgG1conjugated with AlexaFluor647 at 1/100 dilution for CYTX-DP (the APCchannel y-axis in FIG. 19, panels B and D). None of the cellstransformed with plasmids encoding eCLiPS3.0-scFvFS-Nterm oreCLiPS3.0-expressed F5 scFv or anti-CTLA-4 antibody, respectively (FIG.19, Panel A, C). In contrast, F5 scFv and anti-CLTA-4 antibody wereexpressed in all cell types transformed with plasmids encodingCYTX-DP-scFvFS-Nterm or CYTX-DP-antiCTLA4-Nterm, with a range from 8-30%(FIG. 19, Panel B, D).

Example 11 Construction of libraries comprising CYTX-DP platforms withsubstrate or Substrate Library DMs

This Example describes a method to produce a library of the embodimentsthat encodes a CYTX-DP with a DM that is either a substrate or a libraryof substrates.

Vector pB33-GSNS1-GGS4-eCLiPS3.0 PHB4901 was mutated to remove the BglIsite by a single point mutation C→G at nucleotide position 109 using theQuikchange Lightning mutagenesis kit (Agilent Technologies, Santa Clara,Calif., formerly Stratagene). A nucleic acid molecule encodingCYTX-DP-GSNS was sub-cloned into the vector between the BamHI andHindIII sites. The nucleotides encoding the N-terminal substrate site(DM) boundaries of the CYTX-DP were designed to be non-compatible BglIsites in the nucleotide sequence encoding the linker between theN-terminal tag and the transmembrane domain (CYTX-CP) in order to useinexpensive, small oligonucleotide cassettes for small substrates,peptides and libraries and quality controlled cut vector. Into thissubstrate site between the BglI sites, a fragment of the B. subtilisstructural SacB gene, a commonly used molecular biology stuffersequence, was sub-cloned in order to achieve a better quality cutvector; this could also be done by cutting a second time with anotherenzyme. The vector was digested overnight at 37° C. with 50 U BglI (NEB)per 150 ug of vector. Antarctic phosphatase (NEB) was then used for 30minutes at 37° C. to remove phosphate groups from the vector DNAfollowed by heat inactivation at 65° C. for 10 minutes before agarosegel purification. Substrate oligonucleotides or substrate libraryoligonucleotides having top and bottom strands with compatible BglI endsand chemical phosphorylation with standard desalting purification wereordered from standard DNA synthesis vendors (e.g., Elim Biopharm,Fremont Calif. or IDT, Coralville, Iowa). Top and bottomoligonucleotides were mixed in equimolar amounts, annealed with a −0.5°C. gradient from 99° C. to 4° C. using an annealing protocol. Digestedvector and annealed substrate insert DNA were mixed in various ratiosfor ligation and piloted for the best molar ratio. Ligation wasperformed at room temperature overnight; ligase (NEB or Lucigen) washeat inactivated at 70° C. and dialyzed against water. Ligation scale-upwas conducted in a 100 mg ligation reaction for library production andmuch less for single clones. The electroporation protocol for bacteriaused 2 to 5 uL DNA per 25-50 uL bacterial cells thawed on wet ice andusing protocols from the vendor (Lucigen, Middleton, Wis.). Cells wereplated or grown in liquid culture over-night at 37° C. and subsequentlypooled and frozen to −80° C. for long-term storage.

Example 12 Selection of a Known Positive from a Negative Background

To demonstrate the utility of the display platforms (DPs) of thedisclosure for selection of substrates cleaved by proteases using FACS aclone expressing a known substrate (referred to herein as 1204) wasspiked into a background of a known negative (ssNSUB). One round ofselection was performed by cleavage of the mixed population usingrecombinant human uPA (rh uPA) at a final concentration of 100 nM. Thepopulations were then analyzed by FACS and the cells that shifted intothe P3 gate (FIGS. 32A-32D) were sorted into a fresh tube. Cells werethen grown overnight, labeled and analyzed by flow cytometry todetermine the level of enrichment.

Samples A-D (FIGS. 32A-32D) were analyzed for percentage of cells in theP3 gate and the data was plotted (FIG. 33): the data shows that after asingle round of FACS the positive portion of the population is enrichedfrom 1% to 15-20%.

Example 13 Selection of Novel Substrates for MT-SP1

To demonstrate the utility of the display platforms (DPs) of thedisclosure for selection of substrates cleaved by proteases using FACS,a substrate library consisting of eight random amino acids wasconstructed using the CYTX-DP platforms of the disclosure. In someembodiments, the CYTX-DP platform includes an EagI site on theC-terminus prior to the histidine tag. In some embodiments, the CYTX-DPplatform includes a NotI site on the C-terminus prior to the histidinetag. Examples of such DPs include, but are not limited, to DPs havingamino acid sequence SEQ ID NO: 81 shown below or amino acid sequence SEQID NO: 82 shown below, where each member of the substrate library has arandom 8-amino acid sequence in the position indicated XXXXXXXX (SEQ IDNO: 61).

CYTX-DP for Substrate Selection (S-CP CYTX-DP comprising T1-L1-S-L2-CP-L3-T2): S-CYTX-DP with  NotI site:(SEQ ID NO: 81) EYMPMEGGSGQSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGHHHHHHHH S-CYTX-DP with EagI site:(SEQ ID NO: 82) EYMPMEGGSGQSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQTAAGHHHHHHHH

A library comprising a DP having amino acid sequence SEQ ID NO: 82 wasthen screened using three rounds of selection with MT-SP1.

The naïve library was initially prepared for selections by MACS toremove non-expressing members. Round 0 (post-MACS) then underwent threefurther rounds of FACS sorting to select a final pool of substrates(FIG. 34).

90 clones form the final pool of substrates were sequenced and thesequences were aligned using CLC Main Workbench (CLC bio). Using thisalignment amino acid positions were designated for each substraterelative to the arginine residue at the expected site of cleavage(between residues P1 and P1′).

As MT-SP1 substrates have been discovered and published previously usingphage display by Takeuchi et al (J Biol. Chem. 2000) we could directlycompare the frequency of amino acids present at positions P1′, P2, P3and P4, the data is presented in FIG. 35.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

What is claimed is:
 1. A composition comprising: a carrier polypeptide(CP) comprising at least a transmembrane polypeptide portion, whereinthe CP comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 56 and SEQ ID NO: 57, and adisplayed moiety (DM) wherein the CP and the DM are operably linked. 2.The composition of claim 1, wherein the DM is displayed on theN-terminus of the CP.
 3. The composition of claim 1, wherein the DM isdisplayed on the C-terminus of the CP.
 4. The composition of claim 1,wherein the CP comprises the amino acid sequence of SEQ ID NO: 1, anamino acid sequence selected from the group consisting of GQSGQ (SEQ IDNO: 7), GGSGQSGQGG (SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS(SEQ ID NO: 59), GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20),and GSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11) N-terminal to the amino acidsequence of SEQ ID NO: 1, and an amino acid sequence selected from thegroup consisting of GGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12),and GGSGGSSGQTAAG (SEQ ID NO: 83) C-terminal to the amino acid sequenceof SEQ ID NO:
 1. 5. The composition of claim 1, wherein the CP comprisesthe amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:
 58. 6. Thecomposition of claim 1, wherein the CP is operably linked to the DM toform a display platform (DP) having the structural arrangement fromN-terminus to C-terminus: CP-DM or DM-CP, wherein the DP comprises anN-terminus tag, a C-terminus tag or both an N-terminus tag and aC-terminus tag.
 7. The composition of claim 6, wherein the DP comprisesboth an N-terminus tag and a C-terminus tag.
 8. The composition of claim7, wherein the CP comprises the amino acid sequence of SEQ ID NO: 1, andwherein: (i) the N-terminal tag comprises an amino acid sequenceselected from the group consisting of GQSGQ (SEQ ID NO: 7), GGSGQSGQGG(SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS (SEQ ID NO: 59),GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20), andGSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11); or (ii) the C-terminal tagcomprises an amino acid sequence selected from the group consisting ofGGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12), and GGSGGSSGQTAAG(SEQ ID NO: 83); or (iii) the N-terminal tag comprises an amino acidsequence selected from the group consisting of GQSGQ (SEQ ID NO: 7),GGSGQSGQGG (SEQ ID NO: 9), GGSGQSGQ (SEQ ID NO: 22), GGSGQSGQGS (SEQ IDNO: 59), GGSG (SEQ ID NO: 15), GGSGGSGGSGGSG (SEQ ID NO: 20), andGSSGGQGGSGGSGGSGGSGGSA (SEQ ID NO: 11) and the C-terminal tag comprisessequence an amino acid sequence selected from the group consisting ofGGS (SEQ ID NO: 17), GGSGGSSGQAAAG (SEQ ID NO: 12), and GGSGGSSGQTAAG(SEQ ID NO: 83).
 9. The composition of claim 7, wherein N-terminal tagcomprises the amino acid sequence EYMPME (SEQ ID NO: 8).
 10. Thecomposition of claim 7, wherein the C-terminal tag comprises a histidinetag.
 11. The composition of claim 10, wherein the histidine tag is an8-His tag.
 12. The composition of claim 7, wherein the N-terminal tagcomprises the amino acid sequence EYMPME (SEQ ID NO: 8) and theC-terminal tag comprises an 8-His tag.
 13. The composition of claim 7,wherein the DP comprises more than one DM.
 14. The composition of claim13, wherein the DP has the structural arrangement from N-terminus toC-terminus: T1-DM1-DM2-CP-T2, T1-CP-DM1-DM2-T2, T1-DM1-DM2-DM3-CP-T2,T1-CP-DM1-DM2-DM3-T2, or T1-DM1-CP-DM2-T2.
 15. The composition of claim1, wherein the composition comprises one or more linkers.
 16. Thecomposition of claim 6, wherein the DP comprises one or more linkers.17. The composition of claim 1, wherein the DM is selected from thegroup consisting of a substrate, an exosite, a masking moiety, anantibody, a receptor, a ligand, and other proteins, and any combinationsthereof.
 18. The composition of claim 17, wherein the substrate is asubstrate for a protease.
 19. A composition comprising: a replicablebiological entity; and a displayed moiety (DM) displayed on the surfaceof the replicable biological entity, wherein the DM is fused to anextracellular terminus of a circularly permuted bacterial outer membranecarrier protein (CP) to produce a display platform (DP), wherein the CPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 56 and SEQ ID NO:
 57. 20. The composition ofclaim 19, wherein the DM is a substrate for an enzyme.
 21. Thecomposition of claim 19, wherein the DP comprises a tag.
 22. Thecomposition of claim 21, wherein the tag is a fluorescent agent.
 23. Thecomposition of claim 21, wherein the tag is a C-terminal tag.
 24. Thecomposition of claim 21, wherein the tag is an N-terminal tag.
 25. Thecomposition of claim 19, wherein the C-terminus of the CP is exposed onthe surface of the replicable biological entity.
 26. The composition ofclaim 19, wherein the N-terminus of the CP is exposed on the surface ofthe replicable biological entity.
 27. The composition of claim 19,wherein the replicable biological entity is a bacterial cell.
 28. Apeptide display scaffold, comprising a fusion protein comprising aformula selected from the group consisting of:[T1-DM]-carrier protein-[T2] and(2)[T1]-carrier protein-[DM-1-T2]  (1)wherein the carrier protein is a circularly permuted bacterial outermembrane protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 56 and SEQ ID NO: 57; DMcomprises a candidate peptide; and T1 and T2 are first and seconddetectable moieties; and wherein, when the fusion protein is expressedin a cell comprising a cell outer membrane, T1-DM and T2 in formula (1)or T1 and DM-T2 in formula (2) are exposed at an extracellular surfaceof the cell outer membrane and one or both of the N- and C-termini ofthe fusion protein are exposed at the extracellular surface of the cellouter membrane.
 29. The composition of claim 1, wherein the CP comprisesthe amino acid sequence of SEQ ID NO:
 1. 30. The composition of claim 1,wherein the CP comprises the amino acid sequence of SEQ ID NO:
 56. 31.The composition of claim 1, wherein the CP comprises the amino acidsequence of SEQ ID NO:
 57. 32. The composition of claim 19, wherein theCP comprises the amino acid sequence of SEQ ID NO:
 1. 33. Thecomposition of claim 19, wherein the CP comprises the amino acidsequence of SEQ ID NO:
 56. 34. The composition of claim 19, wherein theCP comprises the amino acid sequence of SEQ ID NO:
 57. 35. Thecomposition of claim 28, wherein the carrier protein comprises the aminoacid sequence of SEQ ID NO:
 1. 36. The composition of claim 28, whereinthe carrier protein comprises the amino acid sequence of SEQ ID NO: 56.37. The composition of claim 28, wherein the carrier protein comprisesthe amino acid sequence of SEQ ID NO: 57.